Assay systems for genetic analysis

ABSTRACT

The present invention provides assays systems and methods for detection of chromosomal abnormalities and status of single loci associated with monogenic or polygenic traits in a sample containing nucleic acids from a maternal and a fetal source.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of pending applicationU.S. Ser. No. 13/013,732, filed Jan. 25, 2011, which is a US utilityfiling claiming priority to U.S. Ser. No. 61/371,605, filed Aug. 6,2010, which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the detection and quantification of geneticabnormalities in samples from pregnant females.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Genetic abnormalities account for a wide number of pathologies,including syndromes caused by chromosomal aneuploidy (e.g., Downsyndrome) and those caused by germline mutations resulting in eithermonogenic or polygenic diseases or disorders. Diagnostic methods fordetermining genetic anomalies have become standard techniques foridentifying specific syndromes, diseases and disorders. In particular,prenatal diagnostics have become standard practice in high-riskpopulations to determine the presence or absence of certain disorders.Detection of both gross chromosomal abnormalities, such as trisomies,translocations and large insertions or deletions, and single genetraits, such as single gene mutations or polymorphisms associated withRh blood group status, autosomal dominant or X-linked disorders, orautosomal recessive disorders are useful in detecting actual andpotential pathologies and disorders that may affect a fetus. Forexample, chromosomal abnormalities such as trisomies 13, 18, and 21, theRobertsonian translocation associated with certain forms of Downsyndrome, and larger deletions such as those found on chromosome 22 inDiGeorge syndrome all impact significantly on fetal health.

Similarly, detection of single gene disorders in a fetus, e.g.,mutations in genes causing Tay-Sachs disease, sickle cell anemia, andthalassemia or copy number variants in diseases such as spinal muscularatrophy (SMA), may help parents to make important decisions regardingthe health and care of the child. In addition, genetic status associatedwith blood group system status provide important information formaternal and/or and fetal health, and in many instances such knowledgeprovides an opportunity for intervention to prevent any deleteriousoutcomes in the pregnancy or immediately following birth.

Although conventional technology provides detection methods for thesedifferent genetic abnormalities, it currently requires differenttechniques to interrogate different classes of mutations. Conventionalmethods of prenatal diagnostic testing for chromosomal aneuploidycurrently requires removal of a sample of fetal cells directly from theuterus for genetic analysis, using either chorionic villus sampling(CVS) between 11 and 14 weeks gestation or amniocentesis after 15 weeks.However, these invasive procedures carry a risk of miscarriage of aroundone percent (see Mujezinovic and Alfirevic, Obstet. Gynecol 2007;110:687-614. Current analysis of fetal cells typically involvekaryotyping or fluorescent in situ hybridization (FISH) and do notprovide information about single gene traits, and thus additional testsare required for identification of single gene diseases and disorders.Thus, a mother desiring genetic information on the status of a fetusmust undergo multiple tests to test for various genetic abnormalities.

An assay providing quantification of non-polymorphic factors such asgenetic copy number variations with simultaneous identification ofgenetic polymorphisms or mutations in a maternal sample would be apowerful tool to identify, e.g., potential medical complications in amother and her fetus. The present invention addresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention provides assay systems and related methods fordetection of a copy number variation (CNV) and determination of statusof one or more single genes in a mother and/or fetus. More particularly,the present invention provides assay systems and related methods fordetermining copy number variation of one or more loci and detection ofpolymorphisms in one or more loci in maternal samples comprising genomicmaterial (e.g., cell free DNA) from both maternal and fetal cell freenucleic acids. The present invention utilizes a single assay system thatallows the identification of both genetic copy number variations (CNVs)and single gene polymorphisms, and the ability to distinguish betweenfetal and maternal CNVs and polymorphisms in the maternal sample. Tin aspecific aspect this allows a determination of the fetal status of bothcopy number variations (including chromosomal abnormalities such asaneuploidies, translocations, insertions or deletions) andpolymorphisms, e.g., mutations associated with either dominant orrecessive diseases and/or predispositions.

In one aspect, the assay system utilizes amplification and detection ofselected loci in a maternal sample to identify the presence or absenceof a chromosomal aneuploidy and to determine status of selected genesassociated with monogenic or polygenic traits.

In one specific aspect, the invention provides assay systems thatcomprise a single assay system with the ability to determine 1) presenceor absence of one or more CNV in the maternal sample; and 2)polymorphisms associated with monogenic or polygenic traits. The assaysystems can specifically detect copy number of selected loci andpolymorphisms present in selected loci from a fetal source within thematernal sample, and to distinguish this from the copy number andpolymorphisms present in selected loci from the maternal source in thematernal sample. Thus, in some preferred aspects, the CNV andpolymorphisms are determined for fetal DNA within the maternal sample.Preferably, the loci are analyzed through use of cell free nucleicacids, and preferably the cell free nucleic acids analyzed in the assaysystem are cell free DNA (cfDNA).

In another preferred aspects, the CNV and/or polymorphisms aredetermined for maternal and fetal DNA within the maternal sample theinvention provides assay systems that comprise a single assay systemwith the ability to determine 1) presence or absence of one or more CNVin the maternal sample; and 2) fetal polymorphisms associated withmonogenic or polygenic traits; and 3) maternal status for polymorphismsassociated with associated with monogenic or polygenic traits.

In one aspect, the invention provides an assay system for detection ofthe presence or absence of a copy number variation (CNV) of a genomicregion and presence or absence of one or more fetal polymorphisms in amaternal sample using a single assay, the assay comprising the steps ofintroducing a first set of fixed sequence oligonucleotides to a maternalsample under conditions that allow the fixed oligonucleotides tospecifically hybridize to complementary regions on one or more loci inor associated with a genomic region; introducing a second set of fixedsequence oligonucleotides comprising universal primer regions to thematernal sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions onone or more loci with a putative polymorphism; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe loci corresponding to a genomic region and/or the loci with aputative polymorphism; amplifying the contiguous ligation products tocreate amplification products; and detecting the amplification products.The detection of the amplification products correlates to the copynumber of one or more genomic regions and the presence or absence of apolymorphism in one or more fetal loci in the maternal sample.Preferably, the CNV is determined for one or more fetal loci.

In certain aspects, the fixed oligonucleotides hybridize to adjacentregions in the loci, and the fixed oligonucleotides are directly ligatedduring the ligation step of the assay. In other aspects, the fixedoligonucleotides hybridize to non-adjacent regions in the loci that areseparated by one, a few, or several nucleotides, and the region betweenthe fixed oligonucleotides is used as a template for primer extension toclose the gap between the fixed oligonucleotides prior to the ligationstep.

Thus, in a second aspect, the invention provides an assay system fordetection of the presence or absence of copy number variation (CNV) of agenomic region and presence or absence of one or more fetalpolymorphisms in a maternal sample using a single assay, the assaycomprising the steps of: introducing a first set of fixed sequenceoligonucleotides to a maternal sample under conditions that allow thefixed oligonucleotides to specifically hybridize to complementaryregions on one or more loci in or associated with a genomic region;introducing a second set of fixed sequence oligonucleotides to thematernal sample under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions onone or more loci with a putative polymorphism; extending the regionbetween the first and second hybridized oligonucleotide of at least onefixed sequence oligonucleotide set with a polymerase and dNTPs to createtwo adjacently hybridized fixed sequence oligonucleotides from that set;ligating the hybridized oligonucleotides to create contiguous ligationproducts complementary to the loci corresponding to a genomic regionand/or the loci with a putative polymorphism; amplifying the contiguousligation products using the universal primer regions to createamplification products; and detecting the amplification products. Thedetection of the amplification products correlates to the copy number ofone or more genomic region and the presence or absence of a fetalpolymorphism in one or more loci in the maternal sample.

The fixed sequence oligonucleotides used to interrogate the specificloci comprise universal primer regions that are common to substantiallyall of the fixed sequence oligonucleotides used in a particular assay.This allows substantially all of the ligation products produced in asingle assay to be amplified, isolated and/or analyzed using similarmechanisms (e.g., sequence determination or hybridization). Use of suchuniversal primer regions obviates the need for individuallydistinguishable detectable moieties associated with particular loci oralleles, and provides a more efficient and cost-effective mechanisms formultiplexing interrogation and/or analysis of multiple loci from one ormultiple samples. In a preferred aspect, the universal primer regionsare used in sequence determination of the amplification products. Inanother preferred aspect, the same universal primer regions are used inthe fixed sequence oligonucleotides used for detection of genomicregions and the fixed sequence oligonucleotides used for detection ofpolymorphisms.

Each set of fixed sequence nucleic acids is designed to hybridize to atleast two separate regions in a selected locus. In preferred aspects,two or more separate oligos are used in a set to hybridize to theseregions to provide adjacent nucleic acids complementary to the selectedloci. In some aspects, however, a set can comprise a single probe withtwo or more distinct non-adjacent regions that are complementary to theselected loci (e.g., padlock probes), as described in more detailherein. The sets of fixed sequence oligos can be provided in the assaysequentially or simultaneously in the assay.

The genomic region can be a single locus, or it may be a larger region,up to and including a chromosome. For determination of larger genomicregions, sets of loci may be used to determine the size and in certainaspects the boundaries of such genomic regions for which CNV has beendetected.

In certain aspects, the amplification products of the assay are isolatedprior to detection. Preferably, the amplification products are isolatedas individual molecules prior to detection. These isolated amplificationproducts can optionally be further amplified to create identical copiesof all or a portion of the individual amplification products prior todetection, or further amplified to create identical copies of moleculescomplementary to all or a portion of the individual amplificationproducts prior to detection.

The multiplexed assays of the invention allow the analysis of 5 or more,preferably 10 or more, preferably 16 or more, preferably 20 or more,preferably 30 or more, preferably 32 or more, preferably 40 or more,preferably 48 or more, preferably 50 or more, preferably 60 or more,preferably 70 or more, preferably 80 or more, preferably 90 or more, andmore preferably 96 or more selected loci simultaneously. These selectedloci lay be different loci from a single sample, or they may be locifrom two or more individuals. In the latter case, at least one of thetwo fixed sequence oligonucleotides used for analysis of a selectedlocus may comprise a sample identifier (e.g., a “sample index”) thatwill allow the locus to be associated with a particular sample.Alternatively, a sample index may be added during amplification of theligation product by using a primer comprising the sample index.

Preferably, at least one locus interrogated for CNV in a maternal sampleis different from all loci interrogated for polymorphisms in thematernal sample. In specific aspects, several loci interrogated for CNVin a maternal sample are different from the loci interrogated forpolymorphisms in the maternal sample. In more specific aspects, themajority of loci interrogated for CNV in a maternal sample are differentfrom the loci interrogated for polymorphisms in the maternal sample.

In some aspects of the invention, the fixed sequence oligonucleotideshybridize to adjacent regions on a locus, and ligation of theseoligonucleotides results in a ligation product that joins the two fixedsequence oligonucleotides. In other aspects, the fixed sequenceoligonucleotides hybridize to non-adjacent regions on a locus, and theregions between the fixed sequence oligonucleotides is extended using apolymerase and dNTPs. In preferred aspects, the fixed sequenceoligonucleotides hybridize to non-adjacent regions on a locus, and oneor more bridging oligonucleotides hybridize to the region between andadjacent to the set of fixed sequence oligonucleotides. In certainpreferred aspects, the bridging oligonucleotides used can providecontent information on polymorphisms in the loci as well as informationon frequency of loci for CNV analysis.

In other preferred aspects, the interrogation of these loci utilizesuniversal amplification techniques that allow amplification of multipleloci in a single amplification reaction. The selected nucleic acids fordetection of both the CNV and polymorphisms using the assay system ofthe invention can be amplified using universal amplification methodsfollowing the initial selective amplification from the maternal sample.The use of universal amplification allows multiple nucleic acids regionsfrom a single or multiple samples to be amplified using a single orlimited number of amplification primers, and is especially useful inamplifying multiple selected regions in a single reaction.

Thus, in a preferred aspect of the invention, sequences complementary toprimers for use in universal amplification are introduced to theselected loci during or following selective amplification. Preferablysuch sequences are introduced to the ends of such selected nucleicacids, although they may be introduced in any location that allowsidentification of the amplification product from the universalamplification procedure.

In certain preferred aspects, one or both of the primers used comprise asample index or other identifier. In a specific aspect, a sample indexis included in one or more of the universal primers. The sample index isincorporated into the amplification products, and amplification productsfrom different samples may then be combined. The sample index ispreferably detected concurrently with the detection of the CNV orchromosomal abnormality and the detection of polymorphism such that theCNV and polymorphism may be properly assigned to the sample of origin.

Frequencies of selected loci can be determined for a genomic region ofinterest and compared to the frequencies of loci of one or more othergenomic regions of interest and/or one or more reference genomic regionsto detect potential CNVs based on loci frequencies in the maternalsample.

In the assay systems of the invention, the amplification products areoptionally isolated prior to detection. When isolated, they arepreferably isolated as individual molecules to assist in subsequentdetection. Following isolation, the amplification products can befurther amplified to create identical copies of all or a portion of theindividual amplification products prior to detection. Alternatively, theisolated amplification products can be further amplified to createidentical copies of molecules complementary to all or a portion of theindividual amplification products prior to detection.

Various methods of detection of CNVs can be employed in conjunction withthe detection of the polymorphisms in the assay systems of theinvention. In one general aspect, the assay system employs a method fordetermination of a CNV in one or more loci in a maternal sample,comprising the steps of amplifying one or more selected nucleic acidsfrom a first genomic region of interest in a maternal sample; amplifyingone or more selected nucleic acids from a second locus of interest inthe maternal sample, determining the relative frequency of the selectedloci, comparing the relative frequency of the selected loci, andidentifying the presence or absence of a CNV based on the comparedrelative frequencies of the selected nucleic acids from the first andsecond loci. Preferably, the assay method amplifies two or more selectedloci from different genomic regions, although the loci may be located inthe same general genomic region for confirmation of CNVs arising fromchromosomal abnormalities rather than CNVs from a single locus.

In other aspects, the bridging oligos are degenerate oligos. In otheraspects, the bridging oligos are provided as pools of oligos with randomsequences that comprise substantially every combination for theparticular size of the bridging oligo used with the fixed sequenceoligonucleotides.

Although the aspects of the invention using bridging oligonucleotidesare described primarily using a single bridging oligo, it is envisionedthat multiple bridging oligos that hybridize to adjacent, complementaryregions between fixed sequence oligonucleotides can be used in thedescribed methods.

In one preferred aspect, the invention provides an assay system fordetection of the presence or absence of copy number variation (CNV) of agenomic region and presence or absence of one or more polymorphisms in amaternal sample using a single assay, the assay comprising the steps of:introducing a first set of fixed sequence oligonucleotides comprisinguniversal primer regions to a maternal sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on one or more loci in or associated with agenomic region; introducing a second set of fixed sequenceoligonucleotides comprising universal primer regions to the maternalsample under conditions that allow the fixed sequence oligonucleotidesto specifically hybridize to complementary, non-adjacent regions on oneor more loci with a putative polymorphism; introducing one or morebridging oligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to regions in the locibetween the regions complementary to the fixed sequence oligonucleotidesof the sets; ligating the hybridized oligonucleotides to createcontiguous ligation products complementary to the loci corresponding toa genomic region and/or the loci with a putative polymorphism;amplifying the contiguous ligation products using the universal primerregions to create amplification products; and detecting theamplification products. The detection of the amplification productscorrelates to the copy number of one or more genomic region and thepresence or absence of a polymorphism in one or more loci in thematernal sample. Preferably, the CNV is detected for one or more fetalloci.

In another specific aspect, the invention provides an assay system fordetection of the presence or absence of CNV of a genomic region andpresence or absence of one or more polymorphisms in a maternal sampleusing a single assay, the assay comprising the steps of: introducing afirst set of fixed sequence oligonucleotides to a maternal sample underconditions that allow the fixed sequence oligonucleotides tospecifically hybridize to complementary regions on one or more loci inor associated with a genomic region; introducing a second set of fixedsequence oligonucleotides to the maternal sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on one or more loci with a putative polymorphism;introducing one or more bridging oligonucleotides under conditions thatallow the bridging oligonucleotides to specifically hybridize to regionsin the loci between the regions complementary to the fixed sequenceoligonucleotides of the set; extending the region between at least onefixed sequence oligonucleotide and a bridging oligonucleotide with apolymerase and dNTPs to create adjacently hybridized fixed sequenceoligonucleotides and bridging oligonucleotides; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe loci corresponding to a genomic region and/or the loci with aputative polymorphism; amplifying the contiguous ligation products usingthe universal primer regions to create amplification products; anddetecting the amplification products. The detection of the amplificationproducts correlates to the copy number of one or more genomic region andthe presence or absence of a polymorphism in one or more loci in thematernal sample. Preferably, the CNV is detected for one or more fetalloci.

In preferred aspects of the aspects of the invention using bridgingoligonucleotides, the first and second set of fixed sequenceoligonucleotides are introduced prior to introduction of the bridgingoligonucleotides. More preferably, the unhybridized fixed sequenceoligonucleotides are removed prior to introduction of the bridgingoligonucleotides. In some aspects, the bridging oligonucleotides areintroduced simultaneously with the ligation mixture. In other aspects,the hybridization products of the fixed sequence oligonucleotides andthe locus are isolated prior to introduction of the bridgingoligonucleotides.

In a preferred aspect, the assay system provides highly multiplexed lociinterrogation using one or more common bridging oligonucleotides thatare complementary to regions in two or more interrogated loci. Thus, thenumber of bridging oligonucleotides used in the multiplexed assay systemwill be less than the number of loci interrogated in the assay. Incertain specific aspects, the assay system uses a pool of bridgingoligonucleotides that are each designed to be compatible with two ormore loci interrogated using the assay system of the invention. In theseaspects, the bridging oligonucleotides used in the multiplexed assay arepreferably designed to have a T_(m) in a range of ±5° C., morepreferably in a range of ±2° C.

In certain aspects, the assay system multiplexes loci interrogationusing one or more common bridging oligonucleotides that arecomplementary to regions in two or more interrogated loci. Thus, thenumber of bridging oligonucleotides used in the multiplexed assay systemwill be less than the number of loci interrogated in the assay. Incertain specific aspects, the assay system uses a pool of bridgingoligonucleotides that are each designed to be compatible with two ormore loci interrogated using the assay system of the invention.

In certain aspects, the bridging oligonucleotides are between 2-45nucleotides in length. In a specific aspect, the bridgingoligonucleotides are between 3-9 nucleotides in length. In yet anotherspecific aspect, the bridging oligonucleotides are between 10-30nucleotides in length.

The loci interrogated for CNV can in some instances be indicative of aduplication of a larger genomic region, e.g., all or part of achromosome. Preferably, the assay systems can distinguish the copynumber of these loci between the maternal and fetal sources within amaternal sample.

In such aspects, the invention provides assay systems that comprise asingle assay system with the ability to detect within a maternal samplefrom an individual 1) presence or absence of a chromosomal abnormalityassociated with one or more CNV; and 2) presence or absence of one ormore polymorphisms at one or more selected locus. The presence orabsence of the chromosomal abnormality is preferably detected throughthe use of informative loci that allow the assay system to distinguishbetween nucleic acids from a major source and a minor source.

Thus, in a specific aspect, the invention provides an assay system fordetection of the presence or absence of a fetal chromosomal abnormalityand the presence or absence of one or more fetal polymorphisms using asingle assay, the assay comprising the steps of: introducing a first setof fixed sequence oligonucleotides comprising universal primer regionsto a maternal sample under conditions that allow the fixedoligonucleotides to specifically hybridize to complementary regions onone or more loci with putative polymorphisms; introducing a second setof fixed sequence oligonucleotides comprising universal primer regionsto the maternal sample under conditions that allow the fixedoligonucleotides to specifically hybridize to complementary regions ontwo or more loci from a first chromosome; introducing a third set offixed sequence oligonucleotides comprising universal primer regions tothe maternal sample under conditions that allow the fixedoligonucleotides to specifically hybridize to complementary regions ontwo or more loci from a second chromosome; introducing one or morebridging oligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to regions in the locibetween the regions complementary to the fixed sequence oligonucleotidesof the set; ligating the hybridized oligonucleotides to createcontiguous ligation products complementary to the nucleic acids;amplifying the contiguous ligation products to create amplificationproducts; and detecting the amplification products. The detection of theamplification products correlates to the presence or absence of a fetalchromosomal abnormality and the presence or absence of a fetalpolymorphism in one or more loci in the maternal sample.

In another specific aspect, the invention provides an assay system fordetection of the presence or absence of a fetal chromosomal abnormalityand presence or absence of one or more polymorphisms in fetal loci in amaternal sample using a single assay, the assay comprising the steps ofintroducing a first set of fixed sequence oligonucleotides comprisinguniversal primer regions to a maternal sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on one or more loci with a putative polymorphism;introducing a second set of fixed sequence oligonucleotides comprisinguniversal primer regions to the maternal sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on two or more loci from a first chromosome;introducing a third set of fixed sequence oligonucleotides comprisinguniversal primer regions to the maternal sample under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions on two or more loci from a second chromosome;introducing one or more bridging oligonucleotides under conditions thatallow the bridging oligonucleotides to specifically hybridize to regionsin the loci between the regions complementary to the fixed sequenceoligonucleotides of the set; extending the region between the first andsecond hybridized oligonucleotide of at least one fixed sequenceoligonucleotide set with a polymerase and dNTPs to create two adjacentlyhybridized fixed sequence oligonucleotides from that set; ligating thehybridized oligonucleotides to create contiguous ligation productscomplementary to the nucleic acids; amplifying the contiguous ligationproducts to create amplification products; and detecting theamplification products. The detection of the amplification productscorrelates to the presence or absence of a fetal chromosomal abnormalityand the presence or absence of a polymorphism in one or more loci in thematernal sample.

In some instances, the chromosomal abnormality is associated with geneduplication or loci expansion on a chromosome of interest. In otherinstances, the chromosomal abnormality is associated with atranslocation resulting in the presence of an extra portion of achromosome in the genome. In yet other instances, the chromosomalabnormality is associated with aneuploidy of a chromosome of interest.

Thus, in one aspect, the invention provides an assay system fordetection of the presence or absence of a chromosomal abnormality andthe presence or absence of polymorphisms at one or more loci using asingle assay, the assay comprising the steps of introducing a first setof fixed sequence oligonucleotides to a maternal sample under conditionsthat allow the fixed sequence oligonucleotides to specifically hybridizeto complementary regions in two or more nucleic acids corresponding toinformative loci on two or more chromosomes; introducing a second set offixed sequence oligonucleotides to the maternal sample under conditionsthat allow the fixed oligonucleotides to specifically hybridize toadjacent, complementary regions in nucleic acids indicative of thegenetic status of one or more loci associated with a monogenic orpolygenic trait; ligating the hybridized oligonucleotides to createcontiguous ligation products complementary to the nucleic acids;amplifying the contiguous ligation products to create amplificationproducts; and detecting the amplification products. Detection of theamplification product correlates to the detection of the loci in thematernal sample, and can be used to determine the quantity of lociassociated with the presence or absence of the chromosomal abnormalityand the genetic status of one or more loci in the maternal sample.Detected levels of these nucleic acids can thus be used to determine thepresence or absence of the chromosomal abnormality in the fetus and thestatus of gene associated with a monogenic or polygenic trait in themother and/or the fetus.

In another aspect, the invention provides an assay system for detectionof the presence or absence of a chromosomal abnormality and the geneticstatus of one or more loci in a maternal sample using a single assay,the assay comprising the steps of introducing a first set of fixedsequence oligonucleotides to a maternal sample under conditions thatallow the fixed oligonucleotides to specifically hybridize tocomplementary regions in two or more nucleic acids corresponding toinformative loci on two or more chromosomes; introducing a second set offixed sequence oligonucleotides to the maternal sample under conditionsthat allow the fixed oligonucleotides to specifically hybridize tocomplementary regions in nucleic acids indicative of the genetic statusof one or more loci associated with a monogenic or polygenic trait;introducing one or more bridging oligonucleotides under conditions thatallow the fixed sequence oligonucleotides to specifically hybridize tocomplementary regions in the nucleic acids, wherein one or more bridgingoligonucleotides are complementary to a region of the nucleic acidsbetween and immediately adjacent to the regions complementary to thefixed sequence oligonucleotides of each set; ligating the hybridizedoligonucleotides to create contiguous ligation products complementary tothe nucleic acids; amplifying the contiguous ligation products to createamplification products; and detecting the amplification products.Detection of the amplification product correlates to the detection ofthe loci in the maternal sample, and can be used to determine thequantity of loci associated with the presence or absence of thechromosomal abnormality and the genetic status of one or more loci inthe maternal sample. Detected levels of these nucleic acids can thus beused to determine the presence or absence of a fetal chromosomalabnormality (e.g., an aneuploidy) and the presence or absence of apolymorphism at one or more loci from a fetal source and/or the maternalsource within a maternal sample.

In a specific aspect, the invention provides an assay system fordetecting the presence or absence of a fetal aneuploidy and detectingthe status of one or more genes of the blood group system in a maternalsample using a single assay, comprising the steps of introducing a firstset of fixed sequence oligonucleotides to the maternal sample underconditions that allow the fixed oligonucleotides to specificallyhybridize to complementary regions in two or more informative loci ontwo or more chromosomes; introducing a second set of fixed sequenceoligonucleotides to the maternal sample under conditions that allow thefixed oligonucleotides to specifically hybridize to complementaryregions in nucleic acids indicative of the genetic status of one or moregenes associated with blood group status; introducing one or morebridging oligonucleotides under conditions that allow the fixed sequenceoligonucleotides to specifically hybridize to complementary regions inthe nucleic acids, wherein one or more bridging oligonucleotides arecomplementary to a region of the nucleic acids between and immediatelyadjacent to the regions complementary to the fixed sequenceoligonucleotides of each set; ligating the hybridized oligonucleotidesto create contiguous ligation products complementary to the inucleicacids; amplifying the contiguous ligation products to createamplification products having the sequence of the loci; and detectingthe amplification products.

Detection of the amplification product provides detection of the lociassociated with the presence or absence of an aneuploidy and the statusof one or more genes of the blood group system in the maternal sample.Detected levels of these nucleic acids can be used to determine thepresence or absence of an aneuploidy in the fetus and the status of oneor more genes of the blood group system in the mother and/or the fetus.

In another aspect, the present invention utilizes techniques that allowthe identification of both CNVs and infectious agents in a maternalsample. This may be especially helpful to monitor patients in which theclinical outcome may be compromised by the presence of an infectiousagent. For example, pregnant women have changes in their immune systemand thus may be more susceptible to infection with pathogens that mayhave an adverse effect on the mother and/or fetus. Accordingly, inspecific aspects the invention provides an assay system for detection ofthe presence or absence of genetic copy number variation (CNV) of agenomic region and the presence or absence of an infectious agent in amaternal sample using a single assay, the assay comprising the steps of:introducing a first set of fixed sequence oligonucleotides to a maternalsample under conditions that allow the fixed oligonucleotides tospecifically hybridize to complementary regions on one or more loci inor associated with a genomic region; introducing a second set of fixedsequence oligonucleotides to the maternal sample under conditions thatallow the fixed oligonucleotides to specifically hybridize tocomplementary regions in nucleic acids indicative of an infectiousagent; ligating the hybridized oligonucleotides to create a contiguousligation product complementary to the nucleic acids; amplifying thecontiguous ligation product to create amplification products; anddetecting the amplification products. The detection of the amplificationproducts correlates to copy number of the genomic region, the presenceor absence of a polymorphism at one or more loci, and the presence orabsence of an infectious agent in the maternal sample.

In another specific aspect, the invention provides an assay system fordetection of the presence or absence of copy number variation (CNV) of agenomic region, the presence or absence of one or more polymorphisms,and the presence or absence of an infectious agent in a maternal samplefrom an individual using a single assay, the assay comprising the stepsof: introducing a first set of fixed sequence oligonucleotides to amaternal sample under conditions that allow the fixed oligonucleotidesto specifically hybridize to complementary regions on one or more lociin or associated with a genomic region; introducing a second set offixed sequence oligonucleotides to the maternal sample under conditionsthat allow the fixed oligonucleotides to specifically hybridize tocomplementary regions on one or more loci with a putative polymorphism;introducing a third set of fixed sequence oligonucleotides to thematernal sample under conditions that allow the fixed oligonucleotidesto specifically hybridize to complementary regions in nucleic acidsindicative of an infectious agent; introducing one or more bridgingoligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to complementary regions inthe nucleic acids, wherein one or more bridging oligonucleotides arecomplementary to a region of the nucleic acids between the regionscomplementary to the fixed sequence oligonucleotides of the set;ligating the hybridized oligonucleotides to create a contiguous ligationproduct complementary to the nucleic acids; amplifying the contiguousligation product to create amplification products; and detecting theamplification products. The detection of the amplification productscorrelates to copy number of the genomic region, the presence or absenceof a polymorphism at one or more loci, and the presence or absence of aninfectious agent in the maternal sample.

In preferred aspects, the assay systems for detection of infectiousorganisms further comprise introducing one or more bridgingoligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to complementary regions inthe nucleic acids, wherein the one or more bridging oligonucleotides arecomplementary to a region of the nucleic acids between the regionscomplementary to the fixed sequence oligonucleotides of each set.

Levels of selected loci can be determined for a genomic region ofinterest (e.g., a chromosome or a portion thereof) and compared to thequantities of loci of one or more other genomic regions of interestand/or one or more reference genomic regions to detect potentialaneuploidies based on chromosome frequencies in the maternal sample.

In certain aspects, the fixed sequence oligonucleotides compriseuniversal primer regions that are used in amplification of thecontiguous ligation product. In specific assay systems, the unhybridizedfixed sequence oligonucleotides are removed prior to amplification ofthe contiguous ligation product.

In some specific aspects, the first and second fixed sequenceoligonucleotides are introduced prior to introduction of the bridgingoligonucleotides. In other specific aspects, the one or more bridgedoligonucleotides are introduced to the assay simultaneously with thefirst and second fixed sequence oligonucleotides.

In certain aspects, the amplification products resulting from the fixedsequence oligonucleotides and the bridging oligonucleotides areoptionally isolated following ligation and amplification but beforedetection. The amplification products are preferably isolated asindividual molecules prior to detection. The isolated molecules can alsobe further amplified to create identical copies of all or a portion ofthe individual amplification products, or alternatively to createidentical copies of molecules complementary to all or a portion of theindividual amplification products prior to detection.

In a preferred aspect, the assay system of the invention employs fixedsequence oligonucleotides comprising one or more indices. Theamplification product can be detected using detection of the indicesrather than or in addition to detection of the amplified sequenceitself. In specific aspects, the indices are allele-specific indices,i.e. associated with the presence of certain alleles, and detection ofthe index reflects a particular allele under interrogation. For example,allele-specific indices may correspond to the use of a bridgingoligonucleotide complementary for a specific polymorphism or mutation ina locus.

Various methods of detection of the chromosomal abnormality can beemployed in conjunction with the detection of the selected gene statusin the assay systems of the invention. In one general aspect, the assaysystem employs a method for determination of a copy number variation ina genomic region of interest in a maternal sample, comprising the stepsof enriching for one or more selected loci from a first genomic regionof interest in a maternal sample; enriching for one or more selectedloci from a second genomic region of interest in the maternal sample,determining the relative frequency of the selected regions from thefirst and second genomic regions of interest, comparing the relativefrequency of the selected regions from the first and second genomicregion of interest, and identifying the presence or absence of a copynumber variation based on the compared relative frequencies of theselected regions. Preferably, the assay method enriches for two or moreselected nucleic acids regions from the first and second genomic regionof interest.

In another general aspect, the assay system employs a method fordetermining the presence or absence of a chromosomal abnormalityassociated with CNV in a genomic region, comprising the steps ofamplifying one or more selected loci from a first chromosome of interestin a maternal sample; amplifying one or more selected loci from a secondchromosome of interest in the maternal sample, determining the relativefrequency of the selected regions from the first and second chromosomesof interest, comparing the relative frequency of the selected regionsfrom the first and second chromosomes of interest, and identifying thepresence or absence of an aneuploidy based on the compared relativefrequencies of the selected regions.

In yet another general aspect, the assay system employs a method fordetermination of the presence or absence of an aneuploidy, comprisingthe steps of amplifying two or more selected loci in the cell free DNAcorresponding to a first chromosome of interest in a maternal sample;amplifying two or more selected loci in the cell free DNA correspondingto a second chromosome of interest in the maternal sample, determiningthe relative frequency of the selected regions from the first and secondchromosomes of interest, comparing the relative frequency of theselected regions from the first and second chromosomes of interest, andidentifying the presence or absence of an aneuploidy based on thecompared relative frequencies of the selected regions. In a specificaspect, the loci of the first and second chromosomes are amplified in asingle reaction, and preferably in a single reaction contained within asingle vessel.

The selected nucleic acids for detection of both the chromosomalabnormality and the status of the selected genes analyzed using theassay system of the invention can be amplified using universalamplification methods following the initial selective amplification fromthe maternal sample. or enrichment. Preferably, the assay system detectsthe presence or absence of loci in samples that can be easily obtainedfrom a subject, such as blood, plasma, serum and the like. In onegeneral aspect, the assay system utilizes detection of selected regionsin cfDNA in a maternal sample. In one more specific aspect, the assaysystem utilizes detection of selected regions in cell free DNA in amaternal sample to identify the presence or absence of a chromosomalaneuploidy and the status of one or more loci. Detection of single genestatus can be obtained using direct detection of specific mutations orpolymorphisms in the selected locus corresponding to the gene.Aneuploidy in a genomic region of interest can be determined based ondetection of quantities of selected loci and comparison to thequantities of selected loci from another genomic region of interestand/or to the quantities of selected loci from a reference genomicregion of interest. In a particular aspect, the ratio of the frequenciesof the nucleic acid are compared to a reference mean ratio that has beendetermined for a statistically significant population of genetically“normal” subjects, i.e. subjects that do not have the particular geneticanomaly that is being interrogated in a particular assay system.

In a preferred aspect of the invention, the amplification productscorresponding to the selected nucleic acids are isolated as individualmolecules for analysis of the selected loci. These individualamplification products are isolated from one another, and preferablyphysically isolated (e.g., on a substrate or in individual vessels). Theindividual molecules may be further amplified following isolation tomake multiple, identical copies of the amplification product, a portionthereof, or a nucleic acid complementary to the amplification product ora portion thereof. The detection of the individual molecules or theamplification product may be done through sequencing.

In some aspects, the ligation product is not amplified, but rather isdirectly detected following hybridization, e.g., using single moleculesequencing techniques.

Thus, in a specific aspect, the invention provides an assay system fordetection of the presence or absence of a chromosomal abnormality andthe genetic status of one or more loci in a maternal sample using asingle assay, the assay comprising the steps of: introducing a first setof fixed sequence oligonucleotides to a maternal sample under conditionsthat allow the fixed sequence oligonucleotides to specifically hybridizeto complementary regions in two or more nucleic acids corresponding to aparticular chromosome; introducing a second set of fixed sequenceoligonucleotides to the maternal sample under conditions that allow thefixed sequence oligonucleotides to specifically hybridize tocomplementary regions in nucleic acids indicative of the genetic statusof one or more loci associated with a monogenic or polygenic disease,disorder or predisposition; introducing one or more bridgingoligonucleotides under conditions that allow the bridgingoligonucleotides to specifically hybridize to complementary regions inthe nucleic acids, wherein one or more bridging oligonucleotides arecomplementary to a region of the nucleic acids between and immediatelyadjacent to the regions complementary to the fixed sequenceoligonucleotides of each set; ligating the hybridized oligonucleotidesto create contiguous ligation products complementary to the nucleicacids; amplifying the contiguous ligation products to createamplification products having the sequence of the loci; isolatingindividual amplification products; and analyzing the individualamplification products to determine the sequence of all or part of theindividual amplification products. The analysis of the individualamplification products correlates to the presence or absence of achromosomal abnormality and the genetic status of one or more loci inthe maternal sample. For example, the levels of the nucleic acidscorresponding to a particular chromosome may be compared by also usingnucleic acids corresponding to another chromosome, or they may becompared to reference levels for the chromosome being interrogated.

In a preferred aspect, the individual amplification products areanalyzed through sequence determination. In other aspects, theindividual amplification products are analyzed using hybridizationtechniques.

It is a feature of the present invention that copy number of theselected loci can be detected using non-polymorphic detection methods,i.e., detection methods that are not dependent upon the presence orabsence of a particular polymorphism to identify the selected nucleicacid region. In a preferred aspect, the assay detection systems utilizenon-polymorphic detection methods to “count” the relative numbers ofselected loci present in a maternal sample. These numbers can beutilized to determine if, statistically, a maternal sample is likely tohave a CNV in a genomic region of a maternal sample. Such informationcan be used to identify a particular pathology or genetic disorder, toconfirm a diagnosis or recurrence of a disease or disorder, to determinethe prognosis of a disease or disorder, to assist in determiningpotential treatment options, etc.

In some aspects, the methods for determination of copy number variationused by the assay system measure the relative frequencies of selectedloci from different chromosomes in a sample. The levels of the differentselected loci corresponding to specific chromosomes can be individuallyquantified and compared to determine the presence or absence of achromosomal aneuploidy in a maternal sample. The individually quantifiedregions may undergo a normalization calculation or the data may besubjected to outlier exclusion prior to comparison to determine thepresence or absence of an aneuploidy in a maternal sample.

In other aspects, the relative frequencies of the selected loci are usedto determine a chromosome frequency of the first and second chromosomesof interest, and the presence or absence of an aneuploidy is based onthe compared chromosome frequencies of the first and second chromosomesof interest.

In yet other aspects, the relative frequencies of the selected loci areused to determine a chromosome frequency of a chromosome of interest anda reference chromosome, and the presence or absence of an aneuploidy isbased on the compared chromosome frequencies of the chromosome ofinterest and the reference chromosome.

As the assay system of the invention is preferably configured as ahighly multiplexed system, multiple loci from a single or multiplechromosomes within an individual sample and/or multiple samples can beanalyzed simultaneously. In such multiplexed systems, the samples can beanalyzed separately, or they may be initially pooled into groups of twoor more for analysis of larger numbers of samples. When pooled data isobtained, such data is preferably identified for the different samplesprior to analysis of aneuploidy. In some aspects, however, the pooleddata may be analyzed for potential CNVs, and individual samples from thegroup subsequently analyzed if initial results indicates that apotential aneuploidy is detected within the pooled group.

In certain aspects, the assay systems utilize one or more indices thatprovide information on specific samples. For example, an index can beused in selective or universal amplification that is indicative of asample from which the nucleic acid was amplified.

In one particular aspect, the selected loci are isolated prior todetection. The selected loci can be isolated from the maternal sampleusing any means that selectively isolate the particular nucleic acidspresent in the maternal sample for analysis, e.g., hybridization,amplification or other form of sequence-based isolation of the nucleicacids from the maternal sample. Following isolation, the selectednucleic acids are individually distributed in a suitable detectionformat, e.g., on a microarray or in a flow cell, for determination ofthe sequence and/or relative quantities of each selected nucleic acid inthe maternal sample. The relative quantities of the detected nucleicacids are indicative of the number of copies of chromosomes thatcorrespond to the selected nucleic acids present in the maternal sample.

Following isolation and distribution of the selected nucleic acids in asuitable format, the selected sequences are identified, e.g., throughsequence determination of the selected sequence.

In one specific aspect, the invention provides an assay system fordetection of the presence or absence of a fetal aneuploidy, comprisingthe steps of providing a maternal sample comprising maternal and fetalcfDNA, amplifying two or more selected loci from a first and secondchromosome of interest in the maternal sample, amplifying two or moreselected loci from the first and second chromosome of interest in thematernal sample, determining the relative frequency of the selectedregions from the chromosomes of interest, comparing the relativefrequency of the selected loci from the first and second chromosomes ofinterest, and identifying the presence or absence of a fetal aneuploidybased on the compared relative frequencies of the selected loci.

In some specific aspects, the relative frequencies of the loci from agenomic region are individually calculated, and the relative frequenciesof the individual loci are compared to determine the presence or absenceof a chromosomal abnormality. In other specific aspects, the relativefrequencies of the selected loci are used to determine a chromosomefrequency of a first and second chromosome of interest and a referencechromosome, and the copy number variation for the chromosome or agenomic region of the chromosome is based on the compared chromosomefrequencies of the first and second chromosomes of interest.

In another specific aspect, the invention provides an assay system fordetection of the presence or absence of a fetal aneuploidy, comprisingthe steps of providing a maternal sample, amplifying two or moreselected loci from a chromosome of interest in the maternal sample,amplifying two or more selected loci from a reference chromosome in thematernal sample, determining the relative frequency of the selectedregions from the chromosomes of interest and the reference chromosome,comparing the relative frequency of the selected loci from thechromosomes of interest and the reference chromosome, and identifyingthe presence or absence of a fetal aneuploidy based on the comparedrelative frequencies of the selected loci. In some specific aspects, therelative frequencies of the loci are individually calculated, and therelative frequencies of the individual loci are compared to determinethe presence or absence of a fetal aneuploidy. In other specificaspects, the relative frequencies of the loci are used to determine achromosome frequency of the chromosome of interest and the referencechromosome, and the presence or absence of a fetal aneuploidy is basedon the compared chromosome frequencies of the chromosome of interest andthe reference chromosome.

The maternal sample used for analysis can be obtained or derived fromany sample which contains the nucleic acid of interest to be analyzedusing the assay system of the invention. For example, a maternal samplemay be from any maternal fluid which comprises both maternal and fetalcell free nucleic acids, including but not limited to maternal plasma,maternal serum, or maternal blood. In certain aspects, however, the DNAof interest to be analyzed using the assay system of the inventioncomprises DNA from fetal cells. Such samples can be obtained frommaternal sources such as amniotic fluid, placenta (e.g., the chorionicvilli), and the like.

Although preferably the assay system is used to detect cfDNA in amaternal sample, in certain aspects the DNA of interest to be analyzedusing the assay system of the invention comprises DNA directly from thedifferent cell types rather than from a maternal sample containing DNAfrom the major and minor cell types. Such samples can be obtained fromvarious sources depending upon the target DNA. For example, fetal cellsfor analysis can be derived from samples such as amniotic fluid,placenta (e.g., the chorionic villi), and the like. Samples of donororgans can be obtained in an individual by biopsy. Infectious organismscan be isolated directly from an individual and analyzed followingisolation. DNA can be extracted from cancerous cells or tissues and usedfor analysis.

It is a feature of the invention that the nucleic acids analyzed in theassay system do not require polymorphic differences between the fetaland maternal sequences to determine potential CNVs. It is anotherfeature of the invention that the substantial majority of the nucleicacids isolated from the maternal sample and detected in the assay systemprovide information relevant to the presence and quantity of aparticular chromosome in the maternal sample, i.e. the detected selectednucleic acids are indicative of a particular locus associated with achromosome. This ensures that the majority of nucleic acids analyzed inthe assay system of the invention are informative.

In some aspects, multiple loci are determined for each genomic regionunder interrogation, and the quantity of the selected regions present inthe maternal sample are individually summed to determine the relativefrequency of a locus in a maternal sample. This includes determinationof the frequency of the locus for the combined maternal and fetal DNApresent in the maternal sample. Preferably, the determination does notrequire a distinction between the maternal and fetal DNA, although incertain aspects this information may be obtained in addition to theinformation of relative frequencies in the sample as a whole.

In preferred aspects, selected nucleic acids corresponding to regionsfrom a chromosome are detected and summed to determine the relativefrequency of a chromosome in the maternal sample. Frequencies that arehigher than expected for a locus corresponding to one chromosome whencompared to the quantity of a locus corresponding to another chromosomein the maternal sample are indicative of an aneuploidy. This can be acomparison of chromosomes that each may be a putative aneuploid in thefetus (e.g., chromosomes 18 and 21), where the likelihood of both beinganeuploid is minimal. This can also be a comparison of chromosomes whereone is putatively aneuploid (e.g., chromosome 21) and the other acts asa reference chromosome (e.g. an autosome). In yet other aspects, thecomparison may utilize two or more chromosomes that are putativelyaneuploid and one or more reference chromosomes.

In one aspect, the assay system of the invention analyzes multiplenucleic acids representing selected loci on chromosomes of interest, andthe relative frequency of each selected locus from the sample isanalyzed to determine a relative chromosome frequency for eachparticular chromosome of interest in the sample. The chromosomalfrequency of two or more chromosomes or portions thereof is thencompared to statistically determine whether a chromosomal abnormalityexists.

In another aspect, the assay system of the invention analyzes multiplenucleic acids representing selected loci on chromosomes of interest, andthe relative frequency of each selected nucleic acid from the sample isanalyzed and independently quantified to determine a relative amount foreach selected locus in the sample. The sum of the loci in the sample iscompared to statistically determine whether a CNV exists in a maternalsample.

In another aspect, subsets of loci on each chromosome are analyzed todetermine whether a chromosomal abnormality exists. The loci frequencycan be summed for a particular chromosome, and the summations of theloci used to determine aneuploidy. This aspect of the invention sums thefrequencies of the individual loci in each genomic region and thencompares the sum of the loci on a genomic region of one chromosomeagainst a genomic region of another chromosome to determine whether achromosomal abnormality exists. The subsets of loci can be chosenrandomly but with sufficient numbers of loci to yield a statisticallysignificant result in determining whether a chromosomal abnormalityexists. Multiple analyses of different subsets of loci can be performedwithin a maternal sample to yield more statistical power. In anotheraspect, particular loci can be selected that are known to have lessvariation between maternal samples, or by limiting the data used fordetermination of chromosomal frequency, e.g., by ignoring the data fromloci with very high or very low frequency within a sample.

In a particular aspect, the measured quantities of one or moreparticular loci are normalized to account for differences in lociquantity in the sample. This can be done by normalizing for knownvariation from sources such as the assay system (e.g., temperature,reagent lot differences), underlying biology of the sample (e.g.,nucleic acid content), operator differences, or any other variables.

In certain specific aspects, determining the relative percentage offetal DNA in a maternal sample may be beneficial in performing the assaysystem, as it will provide important information on the relativestatistical presence of loci that may be indicative of fetal aneuploidy.In each maternally-derived sample, the fetus will have approximately 50%of its loci inherited from the mother and 50% of the loci inherited fromthe father when no copy number variant is present for that locus.Determining the non-maternal loci can allow the estimation of fetal DNAin a maternal sample, and thus provide information used to calculate thestatistically significant differences in chromosomal frequencies forchromosomes of interest. Such loci could thus provide two forms ofinformation in the assay—allelic information can be used for determiningthe percent fetal DNA contribution in a maternal sample and a summationof the allelic information can be used to determine the relative overallfrequency of that locus in a maternal sample. The allelic information isnot needed to determine the relative overall frequency of that locus.

Thus, in some specific aspects, the relative fetal contribution ofmaternal DNA at the allele of interest can be compared to thenon-maternal contribution at that allele to determine approximate fetalDNA concentration in the sample. In a particular aspect, the estimationof fetal DNA in a maternal sample is determined at those loci where themother is homozygous at the locus for a given allele and a fetus isheterozygous at the allele. This may be due to the allele beinginherited by the fetus from the father at that locus, or it may be dueto a de novo polymorphism at that locus. In this situation, the fetalDNA amount will be approximately twice the amount of the fetal allelethat is different from the mother. In other specific aspects, therelative quantity of solely paternally-derived sequences (e.g.,Y-chromosome sequences or paternally-specific polymorphisms) can be usedto determine the relative concentration of fetal DNA in a maternalsample.

In aspects utilizing autosomal loci, generally the percent fetal DNAcontribution is determined by comparing one or more genetic variationson the one or more informative loci. In some particular aspects, thesegenetic variations are short tandem repeats. In other particularaspects, these genetic variations are one or more single nucleotidepolymorphisms. In other aspects, the percent fetal DNA contribution in amaternal sample is calculated by detecting methylation differencesbetween loci on fetal DNA and maternal DNA.

The amplified molecules in the assay samples are analyzed to determine afirst number of assay samples which contain the selected nucleic acidand a second number of assay samples which contain a reference nucleicacid.

In a specific aspect, the assay system of the invention can be utilizedto determine if a sample taken from a subject is a maternal samplecontaining fetal DNA, thus acting as a confirmatory test for pregnancyor as a quality control step before determining the presence or absenceof aneuploidy.

In another specific aspect, the assay system of the invention can beutilized to determine if the maternal sample contains more than onefetal genome, thus permitting the detection of multiple fraternalfetuses, for example fraternal twins or triplets.

In another specific aspect, the assay system of the invention can beutilized to detect and quantify DNA contamination.

In another specific aspect, the assay system of the invention can beutilized to determine if one or more fetus in a multiples pregnancy islikely to have an aneuploidy and/or genetic mutation, and whetherfurther confirmatory tests should be undertaken to confirm theidentification of the fetus with the abnormality. For example, the assaysystem of the invention can be used to determine if one of two twins hasa high likelihood of an aneuploidy or disease trait, followed by a moreinvasive technique that can distinguish physically between the fetuses,such as amniocentesis or chorionic villi sampling, to determine theidentification of the affected fetus.

In another specific aspect, the assay system of the invention can beutilized to determine if a fetus has a potential mosaicism in a cellpopulation, and whether further confirmatory tests should be undertakento confirm the identification of mosaicism in the mother and/or fetus.In certain instances, determination of the percent fetal nucleic acidsin a maternal sample could assist in quantification of the estimatedlevel of mosaicism. Mosaicism could be subsequently confirmed usingother testing methods that could distinguish mosaic full or partialaneuploidy in specific cells or tissue.

In another aspect, the oligonucleotides for a given selected nucleicacid can be connected at the non-sequence specific ends such that acircular or unimolecular probe may bind thereto. In this aspect, the 3′end and the 5′ end of the circular probe binds to the selected locus andat least one universal amplification region is present in thenon-selected specific sequence of the circular probe.

In certain aspects, the assay format allows the detection of acombination of abnormalities using different detection mechanismsapplied to the maternal sample. For example, fetal aneuploidy can bedetermined through the identification of selected nucleic acids in amaternal sample, and specific mutations may be detected by sequencedetermination of mutations in one or more identified alleles of a knownlocus. Thus, in specific aspects, sequence determination of a selectednucleic acid can provide information on the number of copies of aparticular locus in a maternal sample as well as the presence of amutation in a fetal allele within the maternal sample.

In specific aspects, the assay system provides mechanisms for detectingchromosomal abnormalities, genetic status of one or more loci associatedwith a monogenic or polygenic trait, and the presence or absence of aninfectious agent in a maternal sample using a single assay. Such assaysare able to provide a comprehensive look at maternal and fetal healthusing a single assay system. This single assay comprises the steps ofintroducing a first set of fixed sequence oligonucleotides to a maternalsample under conditions that allow the fixed oligonucleotides tospecifically hybridize to complementary regions in two or more nucleicacids corresponding to informative loci on two or more chromosomes;introducing a second set of fixed sequence oligonucleotides to thematernal sample under conditions that allow the fixed oligonucleotidesto specifically hybridize to complementary regions on nucleic acidsindicative of the genetic status of one or more loci associated with amonogenic or polygenic trait; introducing a third set of fixed sequenceoligonucleotides to the maternal sample under conditions that allow thefixed oligonucleotides to specifically hybridize to complementaryregions in nucleic acids indicative of an infectious agent; introducingone or more bridging oligonucleotides under conditions that allow thefixed sequence oligonucleotides to specifically hybridize tocomplementary regions in the nucleic acids, wherein one or more bridgingoligonucleotides are complementary to a region of the nucleic acidsbetween and immediately adjacent to the regions complementary to thefixed sequence oligonucleotides of each set; ligating the hybridizedoligonucleotides to create a contiguous ligation product complementaryto the nucleic acids; amplifying the contiguous ligation product tocreate amplification products; and detecting the amplification products.The detection of the amplification products correlates to the presenceor absence of a chromosomal abnormality, the genetic status of one ormore loci associated with a monogenic or polygenic trait and/or thepresence or absence of an infectious agent in the maternal sample.

It is an important feature of the assay that the amplification productsare analyzed directly without the need for enrichment of polymorphicregions from the initial maternal sample. Thus, the current inventionallows detection of both CNV and polymorphisms from a maternal samplewithout an intervening polymorphic enrichment step prior to sequencedetermination of the selected loci.

It is another important feature of the assay that both CNV andpolymorphism detection are determined using a targeted approach ofselected amplification and detection. This allows the majority ofinformation gathered in the assay to be useful for the determination ofthe CNV and/or polymorphism interrogated in the locus of interest, andobviates the need to generate sequence reads that must be aligned with areference sequence.

These and other aspects, features and advantages will be provided inmore detail as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a simplified flow chart of the general steps utilized in theassay systems of the invention.

FIG. 2 illustrates a first general schematic for a ligation-based assaysystem of the invention.

FIG. 3 illustrates a second general schematic for a ligation-based assaysystem of the invention.

FIG. 4 is a third general schematic for a ligation-based assay system ofthe invention.

FIG. 5 illustrates the genotyping performance that was obtained usingone assay system of the invention.

FIG. 6 illustrates the elements used for a detection of aneuploidy andpolymorphism for two cohorts of maternal samples.

FIG. 7 is a summary of patient and sample information and data for asubset of a second cohort of pregnant subjects.

FIG. 8 illustrates the chromosome 21 aneuploidy detection achieved usingone aspect of the invention for a first cohort.

FIG. 9 illustrates the chromosome 18 aneuploidy detection achieved usingone aspect of the invention for a first cohort.

FIG. 10 illustrates the chromosome 21 aneuploidy detection achievedusing one aspect of the invention for a second cohort.

FIG. 11 illustrates the chromosome 18 aneuploidy detection achievedusing one aspect of the invention for a second cohort.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The term “amplified nucleic acid” is any nucleic acid molecule whoseamount has been increased at least two fold by any nucleic acidamplification or replication method performed in vitro as compared toits starting amount in a maternal sample.

The term “amplification product” as used herein refers to the productresulting from an amplification reaction using the contiguous ligationproduct as a template, or the product resulting from an amplificationreaction using a molecule complementary to the contiguous ligationproduct as a template.

The term “chromosomal abnormality” refers to any genetic variation thataffects all or part of a chromosome larger than a single locus. Thegenetic variants may include but not be limited to any copy numbervariant such as duplications or deletions, translocations, inversions,and mutations. Examples of chromosomal abnormalities include, but arenot limited to, Down Syndrome (Trisomy 21), Edwards Syndrome (Trisomy18), Patau Syndrome (Trisomy 13), Klinefelter's Syndrome (XXY), Triple Xsyndrome, XYY syndrome, Trisomy 8, Trisomy 16, Turner Syndrome,Robertsonian translocation, DiGeorge Syndrome and Wolf-HirschhornSyndrome.

The terms “complementary” or “complementarity” are used in reference tonucleic acid molecules (i.e., a sequence of nucleotides) that arerelated by base-pairing rules. Complementary nucleotides are, generally,A and T (or A and U), or C and G. Two single stranded RNA or DNAmolecules are said to be substantially complementary when thenucleotides of one strand, optimally aligned and with appropriatenucleotide insertions or deletions, pair with at least about 90% toabout 95% complementarity, and more preferably from about 98% to about100% complementarity, and even more preferably with 100%complementarity. Alternatively, substantial complementarity exists whenan RNA or DNA strand will hybridize under selective hybridizationconditions to its complement. Selective hybridization conditionsinclude, but are not limited to, stringent hybridization conditions.Stringent hybridization conditions will typically include saltconcentrations of less than about 1 M, more usually less than about 500mM and preferably less than about 200 mM. Hybridization temperatures aregenerally at least about 2° C. to about 6° C. lower than meltingtemperatures (T_(m)).

The term “correction index” refers to nucleotides incorporated intoamplification products that allow for identification and correction ofamplification, sequencing or other experimental errors including thedetection of deletion, substitution, or insertion of one or more basesduring sequencing as well as nucleotide changes that may occur outsideof sequencing such as oligo synthesis, amplification, and any otheraspect of the assay. These correction indices may be stand-alone indicesthat are separate sequences, or they may be embedded within otherindices to assist in confirming accuracy of the experimental techniquesused, e.g., a correction index may be a subset of sequences used foruniversal amplification or a subset of nucleotides of a sample locus.

The term “diagnostic tool” as used herein refers to any composition orassay of the invention used in combination as, for example, in a systemin order to carry out a diagnostic test or assay on a patient sample.

The term “disease trait” refers to a monogenic or polygenic traitassociated with a pathological condition, e.g., a disease, disorder,syndrome or predisposition.

The term “hybridization” generally means the reaction by which thepairing of complementary strands of nucleic acid occurs. DNA is usuallydouble-stranded, and when the strands are separated they willre-hybridize under the appropriate conditions. Hybrids can form betweenDNA-DNA, DNA-RNA or RNA-RNA. They can form between a short strand and along strand containing a region complementary to the short one.Imperfect hybrids can also form, but the more imperfect they are, theless stable they will be (and the less likely to form).

The term “informative locus” as used herein refers to a locus that ishomozygous for the mother and heterozygous for the fetus on a particularchromosome or portion of a chromosome interrogated for purposes ofdetermining a chromosomal abnormality, e.g., aneuploidy. Informativeloci for use in the assay system of the invention include loci used forinterrogation of a reference chromosome as well as loci used forinterrogation of a chromosome that is putatively aneuploid.

The terms “locus” and “loci” as used herein refer to a locus of knownlocation in a genome.

The term “maternal sample” as used herein refers to any sample takenfrom a pregnant mammal which comprises both fetal and maternal cell freegenomic material (e.g., DNA).

Preferably, maternal samples for use in the invention are obtainedthrough relatively non-invasive means, e.g., phlebotomy or otherstandard techniques for extracting peripheral samples from a subject.

The term “melting temperature” or T_(m) is commonly defined as thetemperature at which a population of double-stranded nucleic acidmolecules becomes half dissociated into single strands. The equation forcalculating the T_(m) of nucleic acids is well known in the art. Asindicated by standard references, a simple estimate of the T_(m) valuemay be calculated by the equation: T_(m)=81.5+16.6(log10[Na+])0.41(%[G+C])−675/n−1.0 m, when a nucleic acid is in aqueoussolution having cation concentrations of 0.5 M or less, the (G+C)content is between 30% and 70%, n is the number of bases, and m is thepercentage of base pair mismatches (see, e.g., Sambrook J et al.,Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press (2001)). Other references include more sophisticatedcomputations, which take structural as well as sequence characteristicsinto account for the calculation of T_(m).

“Microarray” or “array” refers to a solid phase support having asurface, preferably but not exclusively a planar or substantially planarsurface, which carries an array of sites containing nucleic acids suchthat each site of the array comprises substantially identical oridentical copies of oligonucleotides or polynucleotides and is spatiallydefined and not overlapping with other member sites of the array; thatis, the sites are spatially discrete. The array or microarray can alsocomprise a non-planar interrogatable structure with a surface such as abead or a well. The oligonucleotides or polynucleotides of the array maybe covalently bound to the solid support, or may be non-covalentlybound. Conventional microarray technology is reviewed in, e.g., Schena,Ed., Microarrays: A Practical Approach, IRL Press, Oxford (2000). “Arrayanalysis”, “analysis by array” or “analysis by microarray” refers toanalysis, such as, e.g., sequence analysis, of one or more biologicalmolecules using a microarray.

The term “monogenic trait” as used herein refers to any trait, normal orpathological, that is associated with a mutation or polymorphism in asingle gene. Such traits include traits associated with a disease,disorder, or predisposition caused by a dysfunction in a single gene.Traits also include non-pathological characteristics (e.g., presence orabsence of cell surface molecules on a specific cell type (e.g., bloodgroup status)).

The term “non-maternal” allele means an allele with a polymorphismand/or mutation that is found in a fetal allele (e.g., an allele with ade novo SNP or mutation) and/or a paternal allele, but which is notfound in the maternal allele.

By “non-polymorphic”, when used with respect to detection of selectedloci, is meant a detection of such locus, which may contain one or morepolymorphisms, but in which the detection is not reliant on detection ofthe specific polymorphism within the region. Thus a selected locus maycontain a polymorphism, but detection of the region using the assaysystem of the invention is based on occurrence of the region rather thanthe presence or absence of a particular polymorphism in that region.

As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, andnucleotide derivatives that confer nuclease resistance on the nucleicacid molecule containing them. The term nucleotide as used herein alsorefers to dideoxyribonucleoside triphosphates (ddNTPs) and theirderivatives. Illustrated examples of dideoxyribonucleoside triphosphatesinclude, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

According to the present invention, a “nucleotide” may be unlabeled ordetectably labeled by well known techniques. Fluorescent labels andtheir attachment to oligonucleotides are described in many reviews,including Haugland, Handbook of Fluorescent Probes and ResearchChemicals, 9th Ed., Molecular Probes, Inc., Eugene Oreg. (2002); Kellerand Manak, DNA Probes, 2nd Ed., Stockton Press, New York (1993);Eckstein, Ed., Oligonucleotides and Analogues: A Practical Approach, IRLPress, Oxford (1991); Wetmur, Critical Reviews in Biochemistry andMolecular Biology, 26:227-259 (1991); and the like. Other methodologiesapplicable to the invention are disclosed in the following sample ofreferences: Fung et al., U.S. Pat. No. 4,757,141; Hobbs, Jr., et al.,U.S. Pat. No. 5,151,507; Cruickshank, U.S. Pat. No. 5,091,519; Menchenet al., U.S. Pat. No. 5,188,934; Begot et al., U.S. Pat. No. 5,366,860;Lee et al., U.S. Pat. No. 5,847,162; Khanna et al., U.S. Pat. No.4,318,846; Lee et al., U.S. Pat. No. 5,800,996; Lee et al., U.S. Pat.No. 5,066,580: Mathies et al., U.S. Pat. No. 5,688,648; and the like.Labeling can also be carried out with quantum dots, as disclosed in thefollowing patents and patent publications: U.S. Pat. Nos. 6,322,901;6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143;5,990,479; 6,207,392; 2002/0045045; and 2003/0017264. Detectable labelsinclude, for example, radioactive isotopes, fluorescent labels,chemiluminescent labels, bioluminescent labels and enzyme labels.Fluorescent labels of nucleotides may include but are not limitedfluorescein, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), CASCADE BLUE® (pyrenyloxytrisulfonic acid),OREGON GREEN™ (2′,7′-difluorofluorescein), TEXAS RED™ (sulforhodamine101 acid chloride), Cyanine and5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluroescently labeled nucleotides include [R6G]dUTP,[TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLinkCy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, ArlingtonHeights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim, Indianapolis, Ind.; and Chromosomee Labeled Nucleotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, CASCADEBLUE®-7-UTP (pyrenyloxytrisulfonic acid-7-UTP), CASCADE BLUE®-7-dUTP(pyrenyloxytrisulfonic acid-7-dUTP), fluorescein-12-UTP,fluorescein-12-dUTP, OREGON GREEN™ 488-5-dUTP(2′,7′-difluorofluorescein-5-dUTP), RHODAMINE GREEN™-5-UTP((5-{2-[4-(aminomethyl)phenyl]-5-(pyridin-4-yl)-1H-i-5-UTP)), RHODAMINEGREEN™-5-dUTP((5-{2-[4-(aminomethyl)phenyl]-5-(pyridin-4-yl)-1H-i-5-dUTP)),tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, TEXASRED™-5-UTP (sulforhodamine 101 acid chloride-5-UTP), TEXAS RED™-5-dUTP(sulforhodamine 101 acid chloride-5-dUTP), and TEXAS RED™-12-dUTP(sulforhodamine 101 acid chloride-12-dUTP) available from MolecularProbes, Eugene, Oreg.

The terms “oligonucleotides” or “oligos” as used herein refer to linearoligomers of natural or modified nucleic acid monomers, includingdeoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptidenucleic acid monomers (PNAs), locked nucleotide acid monomers (LNA), andthe like, or a combination thereof, capable of specifically binding to asingle-stranded polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Usually monomers are linked by phosphodiesterbonds or analogs thereof to form oligonucleotides ranging in size from afew monomeric units, e.g., 8-12, to several tens of monomeric units,e.g., 100-200 or more. Suitable nucleic acid molecules may be preparedby the phosphoramidite method described by Beaucage and Carruthers(Tetrahedron Lett., 22:1859-1862 (1981)), or by the triester methodaccording to Matteucci, et al. (J. Am. Chem. Soc., 103:3185 (1981)),both incorporated herein by reference, or by other chemical methods suchas using a commercial automated oligonucleotide synthesizer.

The term “polygenic trait” as used herein refers to any trait, normal orpathological, that is associated with a mutation or polymorphism in morethan a single gene. Such traits include traits associated with adisease, disorder, syndrome or predisposition caused by a dysfunction intwo or more genes. Traits also include non-pathological characteristicsassociated with the interaction of two or more genes.

As used herein the term “polymerase” refers to an enzyme that linksindividual nucleotides together into a long strand, using another strandas a template. There are two general types of polymerase—DNApolymerases, which synthesize DNA, and RNA polymerases, which synthesizeRNA. Within these two classes, there are numerous sub-types ofpolymerases, depending on what type of nucleic acid can function astemplate and what type of nucleic acid is formed.

As used herein “polymerase chain reaction” or “PCR” refers to atechnique for replicating a specific piece of selected DNA in vitro,even in the presence of excess non-specific DNA. Primers are added tothe selected DNA, where the primers initiate the copying of the selectedDNA using nucleotides and, typically, Taq polymerase or the like. Bycycling the temperature, the selected DNA is repetitively denatured andcopied. A single copy of the selected DNA, even if mixed in with other,random DNA, can be amplified to obtain billions of replicates. Thepolymerase chain reaction can be used to detect and measure very smallamounts of DNA and to create customized pieces of DNA. In someinstances, linear amplification methods may be used as an alternative toPCR.

The term “polymorphism” as used herein refers to any genetic changes orvariants in a locus that may be indicative of that particular loci,including but not limited to single nucleotide polymorphisms (SNPs),methylation differences, short tandem repeats (STRs), and the like.

Generally, a “primer” is an oligonucleotide used to, e.g., prime DNAextension, ligation and/or synthesis, such as in the synthesis step ofthe polymerase chain reaction or in the primer extension techniques usedin certain sequencing reactions. A primer may also be used inhybridization techniques as a means to provide complementarity of alocus to a capture oligonucleotide for detection of a specific nucleicacid region.

The term “research tool” as used herein refers to any composition orassay of the invention used for scientific enquiry, academic orcommercial in nature, including the development of pharmaceutical and/orbiological therapeutics. The research tools of the invention are notintended to be therapeutic or to be subject to regulatory approval;rather, the research tools of the invention are intended to facilitateresearch and aid in such development activities, including anyactivities performed with the intention to produce information tosupport a regulatory submission.

The term “sample index” refers generally to a series of uniquenucleotides (i.e., each sample index is unique to a sample in amultiplexed assay system for analysis of multiple samples). The sampleindex can thus be used to assist in loci identification for multiplexingof different samples in a single reaction vessel, such that each samplecan be identified based on its sample index. In a preferred aspect,there is a unique sample index for each sample in a set of samples, andthe samples are pooled during sequencing. For example, if twelve samplesare pooled into a single sequencing reaction, there are at least twelveunique sample indexes such that each sample is labeled uniquely. Theindex may be combined with any other index to create one index thatprovides information for two properties (e.g., sample-identificationindex, sample-locus index).

The term “selected locus” as used herein refers to one or more locicorresponding to a chromosome or one or more selected loci associatedwith a monogenic and/or polygenic trait. Such selected loci may bedirectly isolated and enriched from the sample for detection, e.g.,based on hybridization and/or other sequence-based techniques, or theymay be amplified using the sample as a template prior to detection ofthe sequence. Loci for use in the assay systems of the present inventionmay be selected on the basis of DNA level variation between individuals,based upon specificity for a particular chromosome, based on CG contentand/or required amplification conditions of the selected loci, or othercharacteristics that will be apparent to one skilled in the art uponreading the present disclosure.

The terms “sequencing”, “sequence determination” and the like as usedherein refers generally to any and all biochemical methods that may beused to determine the order of nucleotide bases in a nucleic acid.

The term “specifically binds”, “specific binding” and the like as usedherein, when referring to a binding partner (e.g., a nucleic acid probeor primer, antibody, etc.) that results in the generation of astatistically significant positive signal under the designated assayconditions. Typically the interaction will subsequently result in adetectable signal that is at least twice the standard deviation of anysignal generated as a result of undesired interactions (background).

The term “status” as used herein in relationship to a gene refers to thesequence status of the alleles of a particular gene, including thecoding regions and the non-coding regions that affect the translationand/or protein expression from that gene. The status of a geneassociated with an autosomal dominant disease such as achondroplasia(e.g., the gene encoding the fibroblast growth factor receptor) orHuntington's disease (e.g., the Huntingtin gene), or for an X-linkeddisease in the case of a male fetus, can be classified as affected i.e.,one allele possesses mutation(s) that is causative of the diseases ordisorder, or non-affected, i.e. both alleles lack such mutations(s). Thestatus of a gene associated with an autosomal recessive disease or amaternal gene associated with an X-linked recessive disorder, may beclassified as affected, i.e., both alleles possess mutation(s) causativeof the diseases or disorder; carrier, i.e. one allele possessesmutation(s) causative of the diseases or disorder; or non-affected, i.e.both alleles lack such mutations(s). The status of a gene may alsoindicate the presence or absence of a particular allele associated witha risk of developing a polygenic disease, e.g., a polymorphism that isprotective against a particular disease or disorder or a polymorphismassociated with an enhanced risk for a particular disease or disorder.

DETAILED DESCRIPTION OF THE INVENTION

The assay systems and methods described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, microarray and sequencing technology, which are within theskill of those who practice in the art. Such conventional techniquesinclude polymer array synthesis, hybridization and ligation ofoligonucleotides, sequencing of oligonucleotides, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,equivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler,Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics:Sequence and Genome Analysis (2004); Sambrook and Russell, CondensedProtocols from Molecular Cloning: A Laboratory Manual (2006); andSambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (allfrom Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4thEd.) W.H. Freeman, New York (1995); Gait, “Oligonucleotide Synthesis: APractical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger,Principles of Biochemistry, 3^(rd) Ed., W. H. Freeman Pub., New York(2000); and Berg et al., Biochemistry, 5th Ed., W.H. Freeman Pub., NewYork (2002), all of which are herein incorporated by reference in theirentirety for all purposes. Before the present compositions, researchtools and methods are described, it is to be understood that thisinvention is not limited to the specific methods, compositions, targetsand uses described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to limit thescope of the present invention, which will be limited only by appendedclaims.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “alocus” refers to one, more than one, or mixtures of such loci, andreference to “an assay” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that eachintervening value between the upper and lower limit of that range—andany other stated or intervening value in that stated range—isencompassed within the invention. Where the stated range includes upperand lower limits, ranges excluding either of those included limits arealso included in the invention.

Unless expressly stated, the terms used herein are intended to have theplain and ordinary meaning as understood by those of ordinary skill inthe art. The following definitions are intended to aid the reader inunderstanding the present invention, but are not intended to vary orotherwise limit the meaning of such terms unless specifically indicated.All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing the formulations and methodologiesthat are described in the publication and which might be used inconnection with the presently described invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The Invention in General

The use of selected loci in the assay methods of the invention providesamplification of loci from chromosomes of interest and/or referencechromosomes for detection of chromosomal abnormalities such asaneuploidies and large insertions or deletions. For example, the mostcommon fetal aneuploidies associated with clinical outcomes in livebirths include chromosomes 13, 18, 21 and the sex chromosomes. Thus, thenucleic acids of interest for use in the assay of the invention areselected to detect aneuploidy of these particular chromosomes.

The sets of fixed sequence nucleic acids are designed to hybridize to atleast two separate regions in a selected nucleic acid region. Inpreferred aspects, two or more separate oligos are used to hybridize tothese regions to provide adjacent nucleic acids complementary to theselected nucleic acid region. In some aspects, however, a single probecan be used which comprises two or more distinct non-adjacent regionsthat are complementary to the selected loci (e.g., padlock probes) asdescribed in more detail herein.

A distinct advantage of the invention is that the selected locicorresponding to copy number variation and/or polymorphisms can befurther analyzed using a variety of detection and quantificationtechniques, including but not limited to hybridization techniques,digital PCR and high throughput sequencing determination techniques.Selection probes can be designed against any number of loci for anychromosome. Although amplification of the mixed sample prior to theidentification and quantification of the selection nucleic acids regionsis not mandatory, limited amplification prior to detection can beperformed, in particular if the initial amounts of nucleic acid arelimited.

FIG. 1 is a simplified flow chart of the general steps utilized in theassay systems of the invention. FIG. 1 shows method 100, where in afirst step 110, a maternal nucleic acid sample is provided for analysis.At step 120, a first set of fixed sequence oligonucleotides areintroduced to and combined with the maternal sample. The maternal samplecan be prepared from virtually any sample as such techniques are knownto those of skill in the art (see, e.g., Tietz Textbood of ClinicalChemistry and Molecular Diagnostics, 4th Ed., Chapter 2, Burtis, C.Ashwood E. and Bruns, D, eds. (2006); Chemical Weapons ConventionChemicals Analysis: Sample Collection, Preparation and AnalyticalMethods, Mesilaakso, M., ed., (2005); Pawliszyn, J., Sampling and SamplePreparation for Field and Laboratory, (2002); Venkatesh Iyengar, G., etal., Element Analysis of Biological Samples: Principles and Practices(1998); Drielak, S., Hot Zone Forensics: Chemical, Biological, andRadiological Evidence Collection (2004); Wells, D., High ThroughputBioanalytical Sample Preparation (Progress in Pharmaceutical andBiomedical Analysis) (2002)), each of which is incorporated byreference). Depending on the type of mixed sample chosen, additionalprocessing and/or purification steps may be performed to obtain nucleicacid fragments of a desired purity or size, using processing methodsincluding but not limited to sonication, nebulization, gel purification,PCR purification systems, nuclease cleavage, or a combination of thesemethods. In a preferred aspect, samples comprising cell-free DNA (cfDNA)are used.

At step 120, a first set of fixed sequence oligonucleotides areintroduced to the mixed nucleic acid sample, under conditions that allowthe first set of fixed sequence oligonucleotides to hybridize toselected loci in the maternal sample. The first set of fixed sequenceoligonucleotides are capable of amplifying the loci and determining copynumber variations and/or chromosomal abnormalities via detection of locifrequency and/or content. The nucleic acid sequences capable ofdetermining copy number variations or chromosomal abnormalities includesequences that allow for identification of chromosomal abnormalitiessuch as duplications or deletions, aneuploidies, translocations, orinversions.

At step 130, a second set of fixed sequence oligonucleotides areintroduced to and combined with the maternal sample and first set offixed sequence oligonucleotides under conditions that allow the secondset of fixed sequence oligonucleotides to hybridize to the maternalsample. The second set of fixed sequence oligonucleotides comprisenucleic acid sequences that are complementary to a selected locus orlocus in the maternal sample, able to detect polymorphisms. Washingsteps optionally may be included between steps 120 and 130, and 130 and140.

Although the invention is described as the two sets of oligos introducedto the maternal sample sequentially, the order of the sets may bereversed from that described in the figures or, in preferred aspects,they can be introduced simultaneously.

At step 140, first and second sets of fixed sequence oligonucleotidesthat have hybridized to adjacent regions of the selected loci in thematernal sample are ligated, and at step 150, the ligatedoligonucleotides are amplified. The ligated and amplifiedoligonucleotides are then detected and analyzed, which allows fordetermination of copy number variations or chromosomal abnormalities andidentification of polymorphisms at step 160.

The sets of fixed sequence nucleic acids are designed to hybridize to atleast two separate regions in a selected locus. In preferred aspects,two or more separate oligos are used to hybridize to these regions toprovide adjacent nucleic acids complementary to the selected locus. Insome aspects, however, a single probe can be used which comprises two ormore distinct non-adjacent regions that are complementary to theselected loci including precircular probes such as so-called “padlockprobes” or “molecular inversion probes (MIPs)”.

The present invention provides an improved assay system over more randomtechniques such as massively parallel sequencing, shotgun sequencing,and the use of random digital PCR which have been used by others todetect copy number variations in maternal samples such as maternalblood. These aforementioned approaches rely upon sequencing of all or astatistically significant population of DNA fragments in a sample,followed by mapping of these fragments or otherwise associating thefragments to their appropriate chromosomes. The identified fragments arethen compared against each other or against some other reference (e.g.,normal chromosomal makeup) to determine CNVs on particular chromosomes.These methods are inherently inefficient as compared to the presentinvention, as the primary chromosomes of interest only constitute aminority of data that is generated from the detection of such DNAfragments in the maternal samples.

The assays of the present invention provide targeted detection ofselected loci, which provides information on both the content of theselected region (i.e., presence of a polymorphic region) and informationon the frequency of the detected region in a sample (with or withoutdetecting any putative polymorphisms in that region). This key featureprovides the ability to detect both copy number of selected regions andthe presence or absence of polymorphisms in a selected region as asingle data set from performance of a multiplexed assay of theinvention.

Techniques that are dependent upon a very broad sampling of DNA in asample provide a very broad coverage of the DNA analyzed, but in factare sampling the DNA contained within a sample on a 1× or less basis(i.e., subsampling). In contrast, the selective amplification used inthe present assays are specifically designed to provide depth ofcoverage of particular loci of interest, and provide a “super-sampling”of such selected loci with an average sequence coverage of preferably 2×or more, more preferably sequence coverage of 100× of more, even morepreferably sequence coverage of 1000× or more of the selected loci(including from fetal sources) present in the initial maternal sample.Thus, the assay systems of the invention provide a more efficient andeconomical use of data, and the substantial majority of ligatedolignucleotides analyzed following amplification (i.e., theamplification products) result in affirmative information about thepresence of selected loci in the maternal sample.

A distinct advantage of the invention is that the ligation productsresulting from the assays corresponding to chromosomal abnormalitiesand/or chromosomal abnormalities and polymorphisms can be analyzed usinga variety of detection and quantification techniques, including but notlimited to hybridization techniques, digital PCR and high throughputsequencing determination techniques.

The assay systems of the invention provide a more efficient andeconomical use of data, and the substantial majority of sequencesanalyzed following sample amplification result in affirmativeinformation about the presence of a particular CNV in the mixed sample.Thus, unlike techniques relying on massively parallel sequencing orrandom digital “counting” of chromosome regions and subsequentidentification of relevant data from such counts, the assay system ofthe invention provides a much more efficient use of data collection thanthe random approaches taught by others in the art.

Assay Methods

The assay systems of the invention utilize a general scheme as describedabove, though many different configurations and variations can beemployed, a few of which are described below and more of which areexemplified in U.S. Ser. No. 61/371,605 filed Aug. 6, 2010, and U.S.Ser. No. 13/013,732, both of which are incorporated by reference hereinin their entirety.

FIG. 2 illustrates a first general schematic for a ligation-based assaysystem of the invention. The fixed sequence oligonucleotides 201, 203comprise universal primer regions 209 and 211, respectively, and regionscomplementary to the selected locus 205 and 207, respectively. However,in addition, the assay system in FIG. 2 employs a sample index region221 on the first fixed sequence oligonucleotide 201. In certain aspects,all or a portion of the sequences of the selected loci are directlydetected using the described techniques, e.g., by sequence determinationor hybridization techniques. In the example of FIG. 2, a sample index isassociated with the first fixed sequence oligonucleotide 201. Thedetection of the indices can identify a sequence from a specific samplein a highly multiplexed assay system.

At step 202, the fixed sequence oligonucleotides 201, 203 are introducedin step 202 to the maternal sample 200 and allowed to specifically bindto the selected locus 215. Following hybridization, the unhybridizedfixed sequence oligonucleotides are preferably separated from theremainder of the genetic sample (by, e.g., washing—not shown). Abridging oligo is then introduced and allowed to hybridize in step 204to the region of the locus 215 between the first 201 and second 203fixed sequence oligonucleotides. The bound oligonucleotides are ligatedat step 206 to create a contiguous nucleic acid spanning andcomplementary to the locus of interest. In certain aspects of theinvention, the bridging oligonucleotides of are between 2-45 nucleotidesin length. In a specific aspect, the bridging oligonucleotides arebetween 3-9 nucleotides in length. In yet another specific aspect, thebridging oligonucleotides are between 10-30 nucleotides in length.

Following ligation, the ligation product is eluted from the gDNAtemplate. Universal primers 217, 219 are introduced in step 208 toamplify the ligated first and second fixed sequence oligonucleotides tocreate 210 amplification products 223 that comprise the sequence of thelocus of interest. These products 223 are isolated, detected, identifiedand quantified to provide information regarding the presence and amountof the selected loci in the maternal sample. Preferably, theamplification products are detected and quantified through sequencedetermination. In specific aspects, it is desirable to determine thesequences of both the index and the amplification products, for example,to provide identification of the sample as well as the locus. Theindices envisioned in the invention may be associated with the firstfixed sequence oligonucleotides, the second fixed sequenceoligonucleotides or both. Alternatively or in addition, indices may beassociated with primers that are used to amplify the ligated first andsecond fixed sequence oligonucleotides, which also serves to incorporateindices into the amplification products.

In preferred aspects, indices representative of the maternal sample fromwhich a nucleic acid may be isolated are used to identify the source ofthe selected loci in a multiplexed assay system. In such aspects, thenucleic acids are uniquely identified with the sample index. Uniquelyidentified oligonucleotides may then be combined into a single reactionvessel with nucleic acids from other maternal samples prior tosequencing. In such a case, the sequencing data is segregated by theunique sample index to determine the frequency of each target locus foreach maternal sample and to determine whether there is a chromosomalabnormality in an individual sample.

In aspects of the invention using sample indices, the fixed sequenceoligonucleotides preferably are designed so that sample indicescomprising identifying information are located between the universalprimer regions 209 and 211 and the regions complementary to the selectedloci in the sample 205 and 207. Alternatively, the indices and universalamplification sequences can be added to the ligated first and secondfixed sequence oligos (and the bridging oligo, if present) by includingthese indices in the primers used to amplify the ligation products forseparate samples. In either case, preferably the indices are encodedupstream of the locus-specific sequences but downstream of the universalprimers so that they are preserved upon amplification.

FIG. 3 exemplifies methods of the assay system in which one or morebridging olignucleotides are employed and exemplifies how polymorphismsmay be detected and identified. In FIG. 3, two fixed sets of sequenceoligonucleotides are used which comprise substantially the sameuniversal primers 309, 311 and sequence-specific regions 305, 307, butcomprise different sample indices, 321, 323 on the fixed sequenceoligonucleotides of the set where the different indices correspond todifferent base sequences for the single nucleotide polymorphism presentin a particular sample. The ligation reactions are carried out withmaterial from the same maternal sample 300, but in separate tubes withthe different allele-specific oligo sets. Bridging oligonucleotidescorresponding to two possible nucleotides for this SNP in the selectedloci 313, 333 are used to detect of the selected locus in each ligationreaction. Two allele indices 321, 323 that are indicative of theparticular polymorphic alleles are incorporated into the amplificationproducts so that sequence determination of the actual sequence of theligated first, second and bridging oligonucleotides are not necessarilyneeded, although the sequences of the entire ligation products may stillbe determined to identify and/or provide confirmation.

Each of the fixed sequence oligonucleotides comprises a regioncomplementary to the selected locus 305, 307, and universal primerregions 309, 311 used to amplify the different selected loci followinginitial selection and/or isolation of the selected loci from thematernal sample. The universal primer regions are located at the ends ofthe fixed sequence oligonucleotides 301, 303, and 323 flanking theindices and the regions complementary to the nucleic acid of interest,thus preserving the nucleic acid-specific sequences and the sampleindices in the products of any universal amplification methods. Thefixed sequence oligonucleotides 301, 303, 323 are introduced at step 302to an aliquot of the genetic sample 300 and allowed to specifically bindto the selected loci 315 or 325. Following hybridization, theunhybridized fixed sequence oligonucleotides are preferably separatedfrom the remainder of the genetic sample by, e.g., washing (not shown).

The bridging oligos corresponding to an A/T SNP 313 or a G/C SNP 333 areintroduced and allowed to bind in step 304 to the region of the selectedlocus 315 or 325 between the first 305 and second 307 nucleicacid-complementary regions of the fixed sequence oligonucleotides.Alternatively, the bridging oligos 313, 333 can be introduced to thesample simultaneously with the fixed sequence oligonucleotides. Thebound oligonucleotides are ligated in step 306 in the single reactionmixture to create a contiguous nucleic acid spanning and complementaryto the selected locus.

Following ligation, the separate reactions may preferably be combinedfor the universal amplification and detection steps. Universal primers317, 319 are introduced to the combined reactions at step 308 to amplifythe ligated template regions and create at step 310 ligated first andsecond fixed sequence oligos and bridging oligo products 327, 329 thatcomprise the sequence of the selected locus representing both SNPs inthe selected locus. These ligation products 327, 329 are detected andquantified through sequence determination of the ligation product,through the sample index and/or the region of the product containing theSNP in the selected locus.

In an alternative configuration of the methods of the assay systems ofthe invention, the bridging oligo may hybridize to a region that is notdirectly adjacent to the region complementary to one or both of thefixed sequence oligos, and an intermediate step requiring extension ofone or more of the oligos is necessary prior to ligation. For example,as illustrated in FIG. 4, each set of oligonucleotides preferablycontains two oligonucleotides 401, 403 of fixed sequence and one or morebridging oligonucleotides 413. Each of the fixed sequenceoligonucleotides comprises a region complementary to the selected locus405, 407, and primer sequences, preferably universal primer sequences,409, 411, i.e., oligo regions complementary to universal primers. Theprimer sequences 409, 411 are located at or near the ends of the fixedsequence oligonucleotides 401, 403, and thus preserve the nucleicacid-specific sequences in the products of any universal amplificationmethods. The fixed sequence oligonucleotides 401, 403 are introduced atstep 402 to the maternal sample 400 and allowed to specifically bind tothe complementary portions of the locus of interest 415. Followinghybridization, the unhybridized fixed sequence oligonucleotides arepreferably separated from the remainder of the genetic sample (notshown).

The bridging oligonucleotide is then introduced at step 404 and allowedto bind to the region of the selected locus 415 between the first 401and second 403 fixed sequence oligonucleotides. Alternatively, thebridging oligo can be introduced simultaneously with the fixed sequenceoligonucleotides. In this exemplary aspect, the bridging oligohybridizes to a region directly adjacent to the first fixed sequenceoligo region 405, but is separated by one or more nucleotides from thecomplementary region of the second fixed sequence oligonucleotide 407.Following hybridization of the fixed sequence and bridging oligos, thebridging oligo 413 is extended at step 406, e.g., using a polymerase anddNTPs, to fill the gap between the bridging oligo 413 and the secondfixed sequence oligo 403. Following extension, the boundoligonucleotides are ligated at step 408 to create a contiguous nucleicacid spanning and complementary to the locus of interest 415. Afterligation, universal primers 417, 419 are introduced at step 410 toamplify the ligated first, second and bridging oligos to create at step412 amplification products 423 that comprise the sequence of theselected locus of interest. Amplification products 423 are optionallyisolated, detected, and quantified to provide information on thepresence and amount of the selected locus(s) in the maternal sample.

Detecting Copy Number Variations

The assay systems utilize nucleic acid probes designed to identify, andpreferably to isolate, selected nucleic acids regions in a maternalsample. Certain of the probes identify sequences of interest in selectedloci interrogated for copy number (i.e. loci frequency), and otherprobes identify sequences that correspond to polymorphisms of interest(i.e. loci content) in nucleic acids corresponding to a fetal ormaternal source in a maternal sample.

In specific aspects, the assay systems of the invention employ one ormore selective amplification steps (e.g., using one or more primers thatspecifically hybridize to a selected locus) for isolating, amplifying oranalyzing substantially all of the selected loci analyzed. This is indirect contrast to the random amplification approach used by othersemploying, e.g., massively parallel sequencing, as such amplificationtechniques generally involve random amplification of all or asubstantial portion of the genome. In addition, although the initialsample can be enriched using methods such as general amplification toincrease the copy number of nucleic acids in the maternal sample,preferably no enrichment steps are used prior to the hybridization,ligation, and amplification steps used to identify the loci of interest.

In a general aspect, the user of the invention analyzes multipleselected loci on different chromosomes. When multiple loci are analyzedfor a sample, a preferred embodiment is to amplify all of the selectedloci for each sample in one reaction vessel. The frequency or amount ofthe multiple selected loci are analyzed to determine whether achromosomal abnormality exists, and the presence or absence of apolymorphism is analyzed to determine the presence or absence orlikelihood calculation of a fetal chromosomal abnormality in a source inthe maternal sample.

In preferred aspects, multiple selected loci from two or more samplesmay be amplified in a single reaction vessel, and the informationsimultaneously analyzed in a single data set, e.g., through sequencedetermination. The resulting data is then analyzed to separate theresults for the different sample and used to determine the presence ofabsence of CNV and/or the presence of absence of polymorphisms forindividual samples.

In one aspect, chromosomal abnormalities are identified in the assaysystem of the invention using multiple selected loci on multiplechromosomes, and the frequency of the selected loci on the multiplechromosomes compared to identify an increase likelihood of aneuploidybased on the ratios of the chromosomes. Normalization or standardizationof the frequencies can be performed for one or more selected loci.

In another aspect, the assay system sums the frequencies of the selectedloci on two or more chromosomes and then compares the sum of theselected loci on one chromosome against another chromosome to determinewhether a chromosomal aneuploidy exists. In another aspect, the assaysystem analyzes subsets of selected loci on two or more chromosomes todetermine whether a chromosomal aneuploidy exists for one of the twochromosomes. The comparison can be made either within the same ordifferent chromosomes.

In certain aspects, the data used to determine the frequency of theselected loci may exclude outlier data that appear to be due toexperimental error, or that have elevated or depressed levels based onan idiopathic genetic bias within a particular sample. In one example,the data used for summation may exclude DNA regions with a particularlyelevated frequency in one or more samples. In another example, the dataused for summation may exclude selected loci that are found in aparticularly low abundance in one or more samples.

In another aspect subsets of loci can be chosen randomly but withsufficient numbers of loci to yield a statistically significant resultin determining whether a chromosomal abnormality exists. Multipleanalyses of different subsets of loci can be performed within a maternalsample to yield more statistical power. For example, if there are 100selected regions for chromosome 21 and 100 selected regions forchromosome 18, a series of analyses could be performed that evaluatefewer than 100 regions for each of the chromosomes. In this example,selected loci are not being selectively excluded.

The quantity of different nucleic acids detectable on certainchromosomes may vary depending upon a number of factors, includinggeneral representation of loci in different cell sources in maternalsamples, degradation rates of the different nucleic acids representingdifferent loci in maternal samples, sample preparation methods, and thelike. Thus, in another aspect, the quantity of particular loci on achromosome is summed to determine the loci quantity for differentchromosomes in the sample. The loci frequencies are summed for aparticular chromosome, and the sum of the loci are used to determineaneuploidy. This aspect of the invention sums the frequencies of theindividual loci on each chromosome and then compares the sum of the locion one chromosome against another chromosome to determine whether achromosomal abnormality exists.

The nucleic acids analyzed using the assay systems of the invention arepreferably selectively amplified and optionally isolated from thematernal sample using primers specific to the locus of interest (e.g.,to a locus of interest in a maternal sample). The primers for suchselective amplification designed to isolate regions may be chosen forvarious reasons, but are preferably designed to 1) efficiently amplify aregion from the chromosome of interest; 2) have a predictable range ofexpression from maternal and/or fetal sources in different maternalsamples; 3) be distinctive to the particular chromosome, i.e., notamplify homologous regions on other chromosomes. The following areexemplary techniques that may be employed in the assay system or theinvention.

The assay system of the invention detects both fetal aneuploidies andspecific chromosomal abnormalities through identification andquantification of specific loci of interest. Such additionalabnormalities include, but are not limited to, deletion mutations,insertion mutations, copy number polymorphisms, copy number variants,chromosome 22q11 deletion syndrome, 11q deletion syndrome on chromosome11, 8p deletion syndrome on chromosome 8, and the like. Generally, atleast two selected nucleic acid sequences present on the same orseparate chromosomes are analyzed, and at least one of the selected lociis associated with the fetal allelic abnormality. The sequences of thetwo selected loci and number of copies of the two selected loci are thencompared to determine whether the chromosomal abnormality is present,and if so, the nature of the abnormality.

While much of the description contained herein describes detectinganeuploidy by counting the abundance of loci on one or more putativeaneuploid chromosomes and the abundance of loci on one or more normalchromosomes, the same techniques may be used to detect copy numbervariations where such copy number variation occurs on only a portion ofa chromosome. In this detection of the copy number variations, multipleloci within the putative copy number variation location are compared tomultiple loci outside of the putative copy number variation location.For instance, one may detect a chromosome 22q11 deletion syndrome in afetus in a maternal sample by selecting two or more nucleic regionswithin the 22q11 deletion and two or more loci outside of the 22q11deletion. The loci outside of the 22q11 deletion may be on anotherregion of Chromosome 22 or may be on a completely different chromosome.The abundance of each loci is determined by the methods described inthis application.

In some aspects a universal amplification may be used for amplifying theloci. In some aspects, the loci for each sample are assayed in a singlereaction in a single vessel. In other aspects, loci from multiplesamples can be assayed in a single reaction in a single vessel.

Certain aspects of the invention can detect a deletion, including theboundaries of such deletions. In some aspects, at least 24 selected locimay be used within the region of the putative deletion and at least 24selected loci may be used outside of the region of the putativedeletion. In another aspect at least 48 selected loci may be used withinthe region of the putative deletion and at least 48 selected loci may beused outside of the region of the putative deletion. In another aspectat least 48 selected loci may be used within the region of the putativedeletion and at least 96 selected loci may be used outside of the regionof the putative deletion. In another aspect at least 48 selected locimay be used within the region of the putative deletion and at least 192selected loci may be used outside of the region of the putativedeletion. In a preferred aspect at least 384 selected loci may be usedwithin the region of the putative deletion and at least 384 selectedloci may be used outside of the region of the putative deletion. Theloci within the deletion are then summed as are the loci outside of thedeletion. These sums are then compared to each other to determine thepresence or absence of a deletion. Optionally, the sums are put into aratio and that ratio may be compared to an average ratio created from anormal population. When the ratio for a sample falls statisticallyoutside of an expected ratio, the deletion is detected. The thresholdfor the detection of a deletion may be twice or more, preferably four ormore times the variation calculated in the normal population.

Polymorphisms Associated with Diseases or Predispositions

The assay systems of the invention are utilized to detect polymorphisms,such as those associated with an autosomal dominant or recessive diseaseor predisposition disorder. Given the multiplexed nature of the assaysystems of the invention, this detection takes place in the same assayas the detection of chromosomal abnormalities in the fetus. Thus asingle assay system can provide diagnostic information on differentclasses of genetic mutations. Accordingly, as the preferred assaysystems of the invention are highly multiplexed and able to interrogatehundreds or even thousands of nucleic acids within a maternal sample, incertain aspects it is desirable to interrogate the sample for nucleicacid markers within the maternal sample, e.g., nucleic acids associatedwith genetic risk or that identify the presence or absence of infectiousorganisms. Thus, the assay systems provide detection of such nucleicacids in conjunction with the detection of nucleic acids for copy numberdetermination within a maternal sample.

Thus, the assay system of the invention can be used to detectpolymorphisms in a maternal sample, where such polymorphisms areassociated with genes associated with autosomal recessive disordersincluding but not limited to blood disorders (e.g., sickle cell anemia,hemophilia or thalassemia), Tay-Sachs, cystic fibrosis, musculardystrophy, Parkinson's disease, Alzheimer's disease and the like;mutations associated with autosomal dominant disorders such asHuntington's disease or achondroplasia; and copy number variationsassociated with single gene disorders (e.g., spinal muscular atrophy).

In other specific aspects, the assay system of the invention can be usedto detect fetal mutations or polymorphisms in a maternal sample, wheresuch mutations or polymorphisms are associated with polygenic disorderssuch as coronary heart disease, diabetes, hypertension, congenital heartdefects, and epilepsy. Examples include mutations in genes associatedwith predispositions such as mutations in cancer susceptibility genes,(e.g. mutations in BRCAI or II or in p53); polymorphisms associated withincreased risk of developing later onset diseases, such as the apoE3gene polymorphism associated with Alzheimer's risk,

In addition to detection of chromosomal abnormalities and single genemutations or polymorphisms associated with monogenic or polygenicdisease, disorders or predispositions, the assay systems of theinvention may identify infectious agents in the maternal sample.Specifically, changes in immunity and physiology during pregnancy maymake pregnant women more susceptible to or more severely affected byinfectious diseases. In fact, pregnancy itself may be a risk factor foracquiring certain infectious diseases, such as toxoplasmosis, Hansendisease, and listeriosis. In addition, for pregnant women or subjectswith suppressed immune systems, certain infectious diseases such asinfluenza and varicella may have a more severe clinical course,increased complication rate, and higher case-fatality rate.Identification of infectious disease agents may therefore allow bettertreatment for maternal disease during pregnancy, leading to a betteroverall outcome for both mother and fetus.

Moreover, certain infectious agents can be passed to the fetus viavertical transmission, i.e. spread of infections from mother to baby.These infections may occur while the fetus is still in the uterus,during labor and delivery, or after delivery (such as whilebreastfeeding).

Thus, is some preferred aspects, the assay system may include detectionof exogenous sequences, e.g., sequences from infectious organisms thatmay have an adverse effect on the health and/or viability of the fetusor infant, in order to protect maternal, fetal, and or infant health.

Exemplary infections which can be spread via vertical transmission, andwhich can be tested for using the assay methods of the invention,include but are not limited to congenital infections, perinatalinfections and postnatal infections.

Congenital infections are passed in utero by crossing the placenta toinfect the fetus. Many infectious microbes can cause congenitalinfections, leading to problems in fetal development or even death.TORCH is an acronym for several of the more common congenitalinfections. These are: toxoplasmosis, other infections (e.g., syphilis,hepatitis B, Coxsackie virus, Epstein-Barr virus, varicella-zoster virus(chicken pox), and human parvovirus B19 (fifth disease)), rubella,cytomegalovirus (CMV), and herpes simplex virus.

Perinatal infections refer to infections that occur as the baby movesthrough an infected birth canal or through contamination with fecalmatter during delivery. These infections can include, but are notlimited to, sexually-transmitted diseases (e.g., gonorrhea, chlamydia,herpes simplex virus, human papilloma virus, etc.) CMV, and Group BStreptococci (GBS).

Infections spread from mother to baby following delivery are known aspostnatal infections. These infections can be spread duringbreastfeeding through infectious microbes found in the mother's breastmilk. Some examples of postnatal infections are CMV, Humanimmunodeficiency virus (HIV), Hepatitis C Virus (HCV), and GBS.

Selected Amplification

Numerous selective amplification methods can be used to provide theamplified nucleic acids that are analyzed in the assay systems of theinvention, and such methods are preferably used to increase the copynumbers of a locus of interest in a maternal sample in a manner thatallows preservation of information concerning the initial content of thelocus in the maternal sample. Although not all combinations ofamplification and analysis are described herein in detail, it is wellwithin the skill of those in the art to utilize different amplificationmethods and/or analytic tools to isolate and/or analyze the nucleicacids of region consistent with this specification, and such variationswill be apparent to one skilled in the art upon reading the presentdisclosure.

Such amplification methods include but are not limited to, polymerasechain reaction (PCR) (U.S. Pat. Nos. 4,683,195; and 4,683,202; PCRTechnology: Principles and Applications for DNA Amplification, ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992), ligase chain reaction (LCR) (Wuand Wallace, Genomics 4:560, 1989; Landegren et al., Science 241:1077,1988), strand displacement amplification (SDA) (U.S. Pat. Nos.5,270,184; and 5,422,252), transcription-mediated amplification (TMA)(U.S. Pat. No. 5,399,491), linked linear amplification (LLA) (U.S. Pat.No. 6,027,923), and the like, self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NASBA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be usedinclude: Qbeta Replicase, described in PCT Patent Application No.PCT/US87/00880, isothermal amplification methods such as SDA, describedin Walker et al., Nucleic Acids Res. 20(7):1691-6 (1992), and rollingcircle amplification, described in U.S. Pat. No. 5,648,245. Otheramplification methods that may be used are described in, U.S. Pat. Nos.5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317 and USPub. No. 20030143599, each of which is incorporated herein by reference.In some aspects DNA is amplified by multiplex locus-specific PCR. In apreferred aspect the DNA is amplified using adaptor-ligation and singleprimer PCR. Other available methods of amplification, such as balancedPCR (Makrigiorgos, et al., Nature Biotechnol, 20:936-9 (2002)) andisothermal amplification methods such as nucleic acid sequence basedamplification (NASBA) and self-sustained sequence replication (Guatelliet al., PNAS USA 87:1874 (1990)). Based on such methodologies, a personskilled in the art readily can design primers in any suitable regions 5′and 3′ to a locus of interest. Such primers may be used to amplify DNAof any length so long that it contains the locus of interest in itssequence.

The length of an amplified selected locus from a genomic region ofinterest is long enough to provide enough sequence information todistinguish it from other nucleic acids that are amplified and/orselected. Generally, an amplified nucleic acid is at least about 16nucleotides in length, and more typically, an amplified nucleic acid isat least about 20 nucleotides in length. In a preferred aspect of theinvention, an amplified nucleic acid is at least about 30 nucleotides inlength. In a more preferred aspect of the invention, an amplifiednucleic acid is at least about 32, 40, 45, 50, or 60 nucleotides inlength. In other aspects of the invention, an amplified nucleic acid canbe about 100, 150 or up to 200 in length.

In certain aspects, the selected amplification comprises an initiallinear amplification step. This can be particularly useful if thestarting amount of DNA from the maternal sample is quite limited, e.g.,where the cell-free DNA in a sample is available in limited quantities.This mechanism increases the amount of DNA molecules that arerepresentative of the original DNA content, and help to reduce samplingerror where accurate quantification of the DNA or a fraction of the DNA(e.g., fetal DNA contribution in a maternal sample) is needed.

Thus, in one aspect, a limited number of cycles of sequence-specificlinear amplification are performed on the starting maternal samplecomprising cfDNA. The number of cycles is generally less than that usedfor a typical PCR amplification, e.g., 5-30 cycles or fewer. Primers orprobes may be designed to amplify specific genomic segments or regions.The primers or probes may be modified with an end label at the 5′ end(e.g. with biotin) or elsewhere along the primer or probe such that theamplification products could be purified or attached to a solidsubstrate (e.g., bead or array) for further isolation or analysis. In apreferred aspect, the primers are multiplexed such that a singlereaction yields multiple DNA fragments from different regions.Amplification products from the linear amplification could then befurther amplified with standard PCR methods or with additional linearamplification.

For example, cfDNA can be isolated from blood, plasma, or serum from apregnant woman, and incubated with primers against a set number of locithat correspond to chromosomes of interest. Preferably, the number ofprimers used for initial linear amplification will be 12 or more, morepreferably 24 or more, more preferably 36 or more, even more preferably48 or more, and even more preferably 96 or more. Each of the primerscorresponds to a single locus, and is optionally tagged foridentification and/or isolation. A limited number of cycles, preferably10 or fewer, are performed with linear amplification. The amplificationproducts are subsequently isolated, e.g., when the primers are linked toa biotin molecule the amplification products can be isolated via bindingto avidin or streptavidin on a solid substrate. The products are thensubjected to further biochemical processes such as further amplificationwith other primers and/or detection techniques such as sequencedetermination and hybridization.

Efficiencies of linear amplification may vary between sites and betweencycles so that in certain systems normalization may be used to ensurethat the products from the linear amplification are representative ofthe nucleic acid content starting material. One practicing the assaysystem of the invention can utilize information from various samples todetermine variation in nucleic acid levels, including variation indifferent loci in individual samples and/or between the same loci indifferent samples following the limited initial linear amplification.Such information can be used in normalization to prevent skewing ofinitial levels of DNA content.

Universal Amplification

In preferred aspects of the invention, the selectively amplified lociare preferably further amplified through universal amplification of allor substantially all of the various loci to be analyzed using the assaysystems of the invention. Universal primer regions are added to thefixed sequence oligonucleotides so that the selectively amplified locimay be further amplified in a single universal amplification reaction.These universal primer sequences may be added to the nucleic acidsregions during the selective amplification process, i.e., the primersfor selective amplification have universal primer sequences that flank alocus. Alternatively, adapters comprising universal amplificationsequences can be added to the ends of the selected nucleic acids asadapters following amplification and isolation of the selected nucleicacids from the maternal sample.

In one exemplary aspect, nucleic acids are initially amplified from amaternal sample using primers complementary to selected regions of thechromosomes of interest, followed by a universal amplification step toincrease the number of loci for analysis. This introduction of primerregions to the initial amplification products from a maternal sampleallows a subsequent controlled universal amplification of all or aportion of selected nucleic acids prior to or during analysis, e.g.sequence determination.

Bias and variability can be introduced during DNA amplification, such asthat seen during polymerase chain reaction (PCR). In cases where anamplification reaction is multiplexed, there is the potential that lociwill amplify at different rates or efficiency. Part of this may be dueto the variety of primers in a multiplex reaction with some havingbetter efficiency (i.e. hybridization) than others, or some workingbetter in specific experimental conditions due to the base composition.Each set of primers for a given locus may behave differently based onsequence context of the primer and template DNA, buffer conditions, andother conditions. A universal DNA amplification for a multiplexed assaysystem will generally introduce less bias and variability.

Accordingly, in a one aspect, a small number (e.g., 1-10, preferably3-5) of cycles of selected amplification or nucleic acid enrichment ofthe initial sample in a multiplexed mixture reaction are performed,followed by universal amplification using introduced universal primers.The number of cycles using universal primers will vary, but willpreferably be at least 10 cycles, more preferably at least 5 cycles,even more preferably 20 cycles or more. By moving to universalamplification following a lower number of amplification cycles, the biasof having certain loci amplify at greater rates than others is reduced.

Optionally, the assay system will include a step between the selectedamplification and universal amplification to remove any excess nucleicacids that are not specifically amplified in the selected amplification.

The whole product or an aliquot of the product from the selectedamplification may be used for the universal amplification. The same ordifferent conditions (e.g., polymerase, buffers, and the like) may beused in the amplification steps, e.g., to ensure that bias andvariability are not inadvertently introduced due to experimentalconditions. In addition, variations in primer concentrations may be usedto effectively limit the number of sequence specific amplificationcycles.

In certain aspects, the universal primer regions of the primers oradapters used in the assay system are designed to be compatible withconventional multiplexed assay methods that utilize general primingmechanisms to analyze large numbers of nucleic acids simultaneously inone reaction in one vessel. Such “universal” priming methods allow forefficient, high volume analysis of the quantity of loci present in amaternal sample, and allow for comprehensive quantification of thepresence of loci within such a maternal sample for the determination ofaneuploidy.

Examples of such assay methods include, but are not limited to,multiplexing methods used to amplify and/or genotype a variety ofsamples simultaneously, such as those described in Oliphant et al., U.S.Pat. No. 7,582,420.

Some aspects utilize coupled reactions for multiplex detection ofnucleic acid sequences where oligonucleotides from an early phase ofeach process contain sequences which may be used by oligonucleotidesfrom a later phase of the process. Exemplary processes for amplifyingand/or detecting nucleic acids in samples can be used, alone or incombination, including but not limited to the methods described below,each of which are incorporated by reference in their entirety.

In certain aspects, the assay system of the invention utilizes one ofthe following combined selective and universal amplification techniques:(1) ligase detection reaction (“LDR”) coupled with polymerase chainreaction (“PCR”); (2) primary PCR coupled to secondary PCR coupled toLDR; and (3) primary PCR coupled to secondary PCR. Each of these aspectsof the invention has particular applicability in detecting certainnucleic acid characteristics. However, each requires the use of coupledreactions for multiplex detection of nucleic acid sequence differenceswhere oligonucleotides from an early phase of each process containsequences which may be used by oligonucleotides from a later phase ofthe process.

Barany et al., U.S. Pat. Nos. 6,852,487, 6,797,470, 6,576,453,6,534,293, 6,506,594, 6,312,892, 6,268,148, 6,054,564, 6,027,889,5,830,711, 5,494,810, describe the use of the ligase chain reaction(LCR) assay for the detection of specific sequences of nucleotides in avariety of nucleic acid samples.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,455,965, 7,429,453,7,364,858, 7,358,048, 7,332,285, 7,320,865, 7,312,039, 7,244,831,7,198,894, 7,166,434, 7,097,980, 7,083,917, 7,014,994, 6,949,370,6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, and6,268,148 describe LDR coupled PCR for nucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,556,924 and 6,858,412, describe the useof precircle probes (also called “padlock probes” or “multi-inversionprobes”) with coupled LDR and polymerase chain reaction (“PCR”) fornucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,709,201, and 7,198, 814describe the use of combined endonuclease cleavage and ligationreactions for the detection of nucleic acid sequences.

Willis et al., U.S. Pat. Nos. 7,700,323 and 6,858,412, describe the useof precircle probes in multiplexed nucleic acid amplification, detectionand genotyping.

Ronaghi et al., U.S. Pat. No. 7,622,281 describes amplificationtechniques for labeling and amplifying a nucleic acid using an adaptercomprising a unique primer and a barcode.

In some cases, a single assay may employ a combination of theabove-described methods. For example, some of the loci may be detectedusing fixed sequence oligonucleotides that bind to adjacent,complementary regions on a locus, while other loci may be detected usingbridging loci in the same assay. In another example, some of the locimay be detected using fixed sequence oligonucleotides that bind toadjacent, complementary regions on a locus, while other loci may requirea primer extension step to join the fixed sequence oligonucleotides.

In a preferred aspect, the amplification products are multiplexed, asdescribed previously. In a preferred aspect, the multiplex amplificationproducts are quantified by analysis of the amplification products. In apreferred aspect, a representational sample of individual molecules fromthe amplification processes is isolated from the other molecules forfurther analysis. To obtain a representational sample of individualmolecules, the average number of molecules per locus must exceed thesampling noise created by the multiplexed reaction. In one aspect, theaverage number per locus is greater than 100. In another aspect, theaverage number per locus is greater than 500. In another aspect theaverage number per locus is greater than 1000.

Individual molecules from the amplification product are preferablyisolated physically from the other molecules in a manner that allows thedifferent amplification products to be distinguished from one another inanalysis. In a preferred aspect, this isolation occurs on a solidsubstrate. The isolated molecule may be associated with a particularidentifiable or physical address either prior to analysis, or theaddress may become known for the particular amplification products basedon the outcome of the analysis. The substrate may be a planar surface orthree-dimensional surface such as a bead.

Once isolated, the individual amplification product may be furtheramplified to make multiple identical copies of that molecule at the sameknown or identifiable location. The amplification may occur before orafter that location becomes an identifiable or physical address. Theamplification product and or its copies (which may be identical orcomplementary to the amplification product) are then analyzed based onthe sequence of the amplification product or its copies to identify theparticular locus and/or allele it represents.

In a preferred aspect, the entire length of the amplification product ora portion of the amplification product may be analyzed using sequencedetermination. The number of bases that need to be determined must besufficient to uniquely identify the amplification product as belongingto a specific locus and/or allele. In one preferred aspect, theamplification product is analyzed through sequence determination of theselected amplification product.

Numerous methods of sequence determination are compatible with the assaysystems of the inventions. Exemplary methods for sequence determinationinclude, but are not limited to, including, but not limited to,hybridization-based methods, such as disclosed in Drmanac, U.S. Pat.Nos. 6,864,052; 6,309,824; and 6,401,267; and Drmanac et al, U.S. patentpublication 2005/0191656, which are incorporated by reference,sequencing by synthesis methods, e.g., Nyren et al, U.S. Pat. Nos.7,648,824, 7,459,311 and 6,210,891; Balasubramanian, U.S. Pat. Nos.7,232,656 and 6,833,246; Quake, U.S. Pat. No. 6,911,345; Li et al, Proc.Natl. Acad. Sci., 100: 414-419 (2003); pyrophosphate sequencing asdescribed in Ronaghi et al., U.S. Pat. Nos. 7,648,824, 7,459,311,6,828,100, and 6,210,891; and ligation-based sequencing determinationmethods, e.g., Drmanac et al., U.S. Pat. Appln No. 20100105052, andChurch et al, U.S. Pat. Appln Nos. 20070207482 and 20090018024.

Sequence information may be determined using methods that determine many(typically thousands to billions) of nucleic acid sequences in anintrinsically parallel manner, where many sequences are read outpreferably in parallel using a high throughput serial process. Suchmethods include but are not limited to pyrosequencing (for example, ascommercialized by 454 Life Sciences, Inc., Branford, Conn.); sequencingby ligation (for example, as commercialized in the SOLiD™ technology,Life Technology, Inc., Carlsbad, Calif.); sequencing by synthesis usingmodified nucleotides (such as commercialized in TruSeq™ and HISEQ™technology by Illumina, Inc., San Diego, Calif., HELISCOPE™ by HelicosBiosciences Corporation, Cambridge, Mass., and PacBio RS by PacificBiosciences of California, Inc., Menlo Park, Calif.), sequencing by iondetection technologies (Ion Torrent, Inc., South San Francisco, Calif.);sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View,Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), and likehighly parallelized sequencing methods.

Alternatively, in another aspect, the entire length of the amplificationproduct or a portion of the amplification product may be analyzed usinghybridization techniques. Methods for conducting polynucleotidehybridization assays for detection of have been well developed in theart. Hybridization assay procedures and conditions will vary dependingon the application and are selected in accordance with the generalbinding methods known including those referred to in: Maniatis et al.Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor,N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide toMolecular Cloning Techniques (Academic Press, Inc., San Diego, Calif.,1987); Young and Davis, P.N.A.S, 80: 1194 (1983). Methods and apparatusfor carrying out repeated and controlled hybridization reactions havebeen described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and6,386,749, 6,391,623 each of which are incorporated herein by reference.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred aspects. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

Variation Minimization within and Between Samples

One challenge with the detection of chromosomal abnormalities in a fetusby detection in a maternal sample is that the nucleic acids from thefetal cell are present in much lower abundance than the nucleic acidsfrom normal cell type. In the case of a maternal sample containing fetaland maternal cell free DNA, the cell free fetal DNA as a percentage ofthe total cell free DNA may vary from less than one to forty percent,and most commonly is present at or below twenty percent and frequentlyat or below ten percent. In the detection of an aneuploidy such asTrisomy 21 (Down Syndrome) in the fetal DNA of such maternal sample, therelative increase in Chromosome 21 is 50% in the fetal DNA and thus as apercentage of the total DNA in a maternal sample where, as an example,the fetal DNA is 5% of the total, the increase in Chromosome 21 as apercentage of the total is 2.5%. If one is to detect this differencerobustly through the methods described herein, the variation in themeasurement of Chromosome 21 has to be much less than the percentincrease of Chromosome 21.

The variation between levels found between samples and/or for lociwithin a sample may be minimized in a combination of analytical methods,many of which are described in this application. For instance, variationis lessened by using an internal reference in the assay. An example ofan internal reference is the use of a chromosome present in a “normal”abundance (e.g., disomy for an autosome) to compare against a chromosomepresent in putatively abnormal abundance, such as aneuploidy, in thesame sample. While the use of one such “normal” chromosome as areference chromosome may be sufficient, it is also possible to use twoor more normal chromosomes as the internal reference chromosomes toincrease the statistical power of the quantification.

One method of using an internal reference is to calculate a ratio ofabundance of the putatively abnormal chromosomes to the abundance of thenormal chromosomes in a sample, called a chromosomal ratio. Incalculating the chromosomal ratio, the abundance or counts of each ofthe loci for each chromosome are summed together to calculate the totalcounts for each chromosome. The total counts for one chromosome are thendivided by the total counts for a different chromosome to create achromosomal ratio for those two chromosomes.

Alternatively, a chromosomal ratio for each chromosome may be calculatedby first summing the counts of each of the loci for each chromosome, andthen dividing the sum for one chromosome by the total sum for two ormore chromosomes. Once calculated, the chromosomal ratio is thencompared to the average chromosomal ratio from a normal population.

The average may be the mean, median, mode or other average, with orwithout normalization and exclusion of outlier data. In a preferredaspect, the mean is used. In developing the data set for the chromosomalratio from the normal population, the normal variation of the measuredchromosomes is calculated. This variation may be expressed a number ofways, most typically as the coefficient of variation, or CV. When thechromosomal ratio from the sample is compared to the average chromosomalratio from a normal population, if the chromosomal ratio for the samplefalls statistically outside of the average chromosomal ratio for thenormal population, the sample contains an aneuploidy. The criteria forsetting the statistical threshold to declare an aneuploidy depend uponthe variation in the measurement of the chromosomal ratio and theacceptable false positive and false negative rates for the desiredassay. In general, this threshold may be a multiple of the variationobserved in the chromosomal ratio. In one example, this threshold isthree or more times the variation of the chromosomal ratio. In anotherexample, it is four or more times the variation of the chromosomalratio. In another example it is five or more times the variation of thechromosomal ratio. In another example it is six or more times thevariation of the chromosomal ratio. In the example above, thechromosomal ratio is determined by summing the counts of loci bychromosome. Typically, the same number of selected loci for eachchromosome is used. An alternative method for generating the chromosomalratio would be to calculate the average counts for the loci for eachchromosome. The average may be any estimate of the mean, median or mode,although typically an average is used. The average may be the mean ofall counts or some variation such as a trimmed or weighted average. Oncethe average counts for each chromosome have been calculated, the averagecounts for each chromosome may be divided by the other to obtain achromosomal ratio between two chromosomes, the average counts for eachchromosome may be divided by the sum of the averages for all measuredchromosomes to obtain a chromosomal ratio for each chromosome asdescribed above. As highlighted above, the ability to detect ananeuploidy in a maternal sample where the putative DNA is in lowrelative abundance depends greatly on the variation in the measurementsof different selected loci in the assay. Numerous analytical methods canbe used which reduce this variation and thus improve the sensitivity ofthis method to detect aneuploidy. One method for reducing variability ofthe assay is to increase the number of selected loci used to calculatethe abundance of the chromosomes. In general, if the measured variationof a single selected locus of a chromosome is X % and Y differentselected loci are measured on the same chromosome, the variation of themeasurement of the chromosomal abundance calculated by summing oraveraging the abundance of each selected locus on that chromosome willbe approximately X % divided by Y^½. Stated differently, the variationof the measurement of the chromosome abundance would be approximatelythe average variation of the measurement of each selected locus'abundance divided by the square root of the number of loci.

In a preferred aspect of this invention, the number of loci measured foreach chromosome is at least 24. In another preferred aspect of thisinvention, the number of selected loci measured for each chromosome isat least 48. In another preferred aspect of this invention, the numberof selected loci measured for each chromosome is at least 100. Inanother preferred aspect of this invention the number of selected locimeasured for each chromosome is at least 200. There is incremental costto measuring each locus and thus it is important to minimize the numberof each selected locus. In a preferred aspect of this invention, thenumber of selected loci measured for each chromosome is less than 2000.In a preferred aspect of this invention, the number of selected locimeasured for each chromosome is less than 1000. In a most preferredaspect of this invention, the number of selected loci measured for eachchromosome is at least 48 and less than 1000. In one aspect, followingthe measurement of abundance for each selected locus, a subset of theselected loci may be used to determine the presence or absence ofaneuploidy. There are many standard methods for choosing the subset ofselected loci. These methods include outlier exclusion, where theselected loci with detected levels below and/or above a certainpercentile are discarded from the analysis. In one aspect, thepercentile may be the lowest and highest 5% as measured by abundance. Inanother aspect, the percentile may be the lowest and highest 10% asmeasured by abundance. In another aspect, the percentile may be thelowest and highest 25% as measured by abundance.

Another method for choosing the subset of selected loci includes theelimination of regions that fall outside of some statistical limit. Forinstance, selected loci that fall outside of one or more standarddeviations of the mean abundance may be removed from the analysis.Another method for choosing the subset of selected loci may be tocompare the relative abundance of a selected locus to the expectedabundance of the same selected locus in a healthy population and discardany selected loci that fail the expectation test. To further minimizethe variation in the assay, the number of times each selected locus ismeasured may be increased. As discussed, in contrast to the randommethods of detecting aneuploidy where the genome is measured on averageless than once, the assay systems of the present invention intentionallymeasures each selected locus multiple times. In general, when countingevents, the variation in the counting is determined by Poissonstatistics, and the counting variation is typically equal to one dividedby the square root of the number of counts. In a preferred aspect of theinvention, the selected loci are each measured on average at least 100times. In a preferred aspect to the invention, the selected loci areeach measured on average at least 500 times. In a preferred aspect tothe invention, the selected loci are each measured on average at least1000 times. In a preferred aspect to the invention, the selected lociare each measured on average at least 2000 times. In a preferred aspectto the invention, the selected loci are each measured on average atleast 5000 times.

In another aspect, subsets of loci can be chosen randomly but withsufficient numbers of loci to yield a statistically significant resultin determining whether a chromosomal abnormality exists. Multipleanalyses of different subsets of loci can be performed within a maternalsample to yield more statistical power. In this example, it may or maynot be necessary to remove or eliminate any loci prior to the randomanalysis. For example, if there are 100 selected loci for chromosome 21and 100 selected loci for chromosome 18, a series of analyses could beperformed that evaluate fewer than 100 loci for each of the chromosomes.

In addition to the methods above for reducing variation in the assay,other analytical techniques, many of which are described earlier in thisapplication, may be used in combination. In general, the variation inthe assay may be reduced when all of the loci for each sample areinterrogated in a single reaction in a single vessel. Similarly, thevariation in the assay may be reduced when a universal amplificationsystem is used. Furthermore, the variation of the assay may be reducedwhen the number of cycles of amplification is limited.

Determination of Fetal DNA Content in Maternal Sample

In certain specific aspects, determining the relative percentage offetal DNA in a maternal sample may be beneficial in performing theassays, as it will provide important information on the expectedstatistical presence of chromosomes and variation from that expectationmay be indicative of fetal aneuploidy. This may be especially helpful incircumstances where the level of fetal DNA in a maternal sample is low,as the percent fetal contribution can be used in determining thequantitative statistical significance in the variations of levels ofidentified loci in a maternal sample. In other aspects, the determiningof the relative percent fetal cell free DNA in a maternal sample may bebeneficial in estimating the level of certainty or power in detecting afetal aneuploidy.

In some specific aspects, the relative fetal contribution of maternalDNA at the allele of interest can be compared to the paternalcontribution at that allele to determine approximate fetal DNAconcentration in the sample. In other specific aspects, the relativequantity of solely paternally-derived sequences (e.g., Y-chromosomesequences or paternally-specific polymorphisms) can be used to determinethe relative concentration of fetal DNA in a maternal sample.

Another exemplary approach to determining the percent fetal contributionin a maternal sample through the analysis of DNA fragments withdifferent patterns of DNA methylation between fetal and maternal DNA. Ina preferred aspect, the amplified DNA from plasma free DNA is bypolymerase chain reaction (PCR). Other mechanisms for amplification canbe used as well, including those described in more detail herein, aswill be apparent to one skilled in the art upon reading the presentdisclosure.

In particular aspects, the percentage of free fetal DNA in the maternalsample can determined by PCR using serially diluted DNA isolated fromthe maternal sample, which can accurately quantify the number of genomescomprising the amplified genes.

In circumstances where the fetus is male, percent fetal DNA in a samplecan be determined through detection of Y-specific nucleic acids andcomparison to calculated maternal DNA content. Quantities of anamplified Y-specific nucleic acid, such as a region from thesex-determining region Y gene (SRY), which is located on the Ychromosome and is thus representative of fetal DNA, can be determinedfrom the sample and compared to one or more amplified genes which arepresent in both maternal DNA and fetal DNA and which are preferably notfrom a chromosome believed to potentially be aneuploid in the fetus,e.g., an autosomal region that is not on chromosome 21 or 18.Preferably, this amplification step is performed in parallel with theselective amplification step, although it may be performed either beforeor after the selective amplification depending on the nature of themultiplexed assay.

PCR using serially diluted DNA isolated from the maternal sample may bepreferred when determining percent fetal DNA with a male fetus. Forexample, if the blood sample contains 100% male fetal DNA, and 1:2serial dilutions are performed, then on average the Y-linked signal willdisappear 1 dilution before the autosomal signal, since there is 1 copyof the Y-linked gene and 2 copies of the autosomal gene.

In a specific aspect, the percentage of free fetal DNA in maternalplasma is calculated for a male fetus using the following formula:percentage of free fetal DNA=(No. of copies of Y-linked gene×2×100)/(No.of copies of autosomal gene), where the number of copies of each gene isdetermined by observing the highest serial dilution in which the genewas detected. The formula contains a multiplication factor of 2, whichis used to normalize for the fact that there is only 1 copy of theY-linked gene compared to two copies of the autosomal gene in eachgenome, fetal or maternal.

Detection of Loci Associated with Pathological Conditions

The assay systems of the invention can be used to identify any lociassociated with a disease trait. This includes loci associated withautosomal recessive diseases and disorders, sex linked diseases anddisorders, and dominant diseases and disorders.

Autosomal Dominant Disease Traits

A disease trait that is inherited in an autosomal dominant manner canoccur in either sex and can be transmitted by either parent. Exemplarydiseases that are inherited in an autosomal dominant fashion include,but are not limited to, achondroplasia, Huntington's disease, Familialhypercholesterolemia, Neurofibromatosis Type I, Hereditaryspherocytosis, and Marfan syndrome. In addition, many of the cancerpredisposition diseases, such as mutations in Rb, p53, and BRCA I and IIare inherited in an autosomal dominant fashion.

Autosomal Recessive Disease Traits

Nearly 2000 genes have been identified that are associated withautosomal recessive diseases. Examples of detectable genetic diseasesinclude, but are not limited to, 21 hydroxylase deficiency, cysticfibrosis, phenylketonuria and other inborn errors in metabolism, sicklecell anemia, Tay-Sachs Syndrome, Roberts syndrome, β-thalassemia,albinism, adrenal hyperplasia, Fanconi anemia, spinal muscularatrophy,myotonic dystrophy, Angelman syndrome, RhD Syndrome, tuberous sclerosis,Mucopolysaccharidoses, Galactosemia, Glycogen storage diseases,Ataxia-telangiectasia, and Prader-Willi syndrome.

X-Linked Disease Traits

In humans, there are hundreds of genes located on the X chromosome thathave no counterpart on the Y chromosome, and the traits governed bythese genes thus display X-linked inheritance. Most sex-linked traitsare recessively inherited, so that heterozygous females generally do notdisplay the trait. The maternal carrier female (heterozygote) has a 50percent chance of passing the mutant gene to each of her children, andso sons who inherit the mutant gene will be hemizygotes and willmanifest the trait, while daughters who receive the mutant gene will beunaffected carriers. Examples of sex-linked disease traits include, butare not limited to, Hemophilia A, Hemophilia B, Duchenne musculardystrophy, Becker's muscular dystrophy, X-linked ichthyosis, X-linkedagammaglobulinemia (XLA), and color blindness.

Non-Mendelian Inherited Disease Traits

Although disorders resulting from single-gene defects that demonstrateMendelian inheritance are perhaps better understood, it is now clearthat a significant number of single-gene diseases also exhibitdistinctly non-Mendelian patterns of inheritance. Among these are suchdisorders that result from triplet repeat expansions within or nearspecific genes (e.g., Huntington disease and fragile-X syndrome); acollection of neurodegenerative disorders, such as Leber hereditaryoptic neuropathy (LHON), that result from inherited mutations in themitochondrial DNA; and diseases that result from mutations in imprintedgenes (e.g., Angelman syndrome and Prader-Willi syndrome).

Blood Group System Traits

In certain preferred aspects of the invention, the assay systems areused to detect a fetal chromosomal abnormality and fetal status of oneor more genes of the human blood group systems. The InternationalSociety of Blood Transfusion (ISBT) currently recognizes 30 major bloodgroup systems (including the ABO and Rh systems). Thus, in addition tothe ABO antigens and Rhesus antigens, many other antigens are expressedon the red blood cell surface membrane. For example, an individual canbe AB RhD positive, and at the same time M and N positive (MNS system),K positive (Kell system), and Le^(a) or Le^(b) positive (Lewis system).Many of the blood group systems were named after the patients in whomthe corresponding antibodies were initially encountered.

The ISBT definition of a blood group system is where one or moreantigens are controlled at a single gene locus or by two or more veryclosely linked homologous genes with little or no observablerecombination between them. See, e.g., ISBT Committee on Terminology forRed Cell Surface Antigens, “Terminology Home Page”. A summary of theblood group systems known at the present time are summarized below inTable 1:

TABLE 1 Human Blood Group Systems ISBT System No. System name symbolEpitope type Chromosome 001 ABO ABO Carbohydrate (N-Acetylgalactosamine,9 galactose). A, B and H antigens. 002 MNS MNS GPA/GPB (glycophorins Aand B). Main 4 antigens M, N, S, s. 003 P P1 Glycolipid. Antigen P1. 22004 Rh RH Protein. C, c, D, E, e antigens (there is no “d” 1 antigen;lowercase “d” indicates the absence of D). 005 Lutheran LU Protein(member of the immunoglobulin 19 superfamily). Set of 21 antigens. 006Kell KEL Glycoprotein. Kell-1 (K₁) 7 007 Lewis LE Carbohydrate (fucoseresidue). Main antigens Le^(a) 19 and Le^(b)—associated with tissue ABHantigen secretion. 008 Duffy FY Protein (chemokine receptor). Mainantigens Fy^(a) 1 and Fy^(b). 009 Kidd JK Protein (urea transporter).Main antigens Jk^(a) and 18 Jk^(b). 010 Diego DI Glycoprotein (band 3,AE 1, or anion exchange). 17 011 Yt or Cartwright YT Protein (AChE,acetylcholinesterase). 7 012 XG XG Glycoprotein. X 013 Scianna SCGlycoprotein. 1 014 Dombrock DO Glycoprotein (fixed to cell membrane byGPI, or 12 glycosyl-phosphatidyl-inositol). 015 Colton CO Aquaporin 1.Main antigens Co(a) and Co(b). 7 016 Landsteiner- LW Protein (member ofthe immunoglobulin 19 Wiener superfamily). 017 Chido/Rodgers CH/RG C4AC4B (complement fractions). 6 018 Hh/Bombay H Carbohydrate (fucoseresidue). 19 019 Kx XK Glycoprotein. X 020 Gerbich GE GPC/GPD(Glycophorins C and D). 2 021 Cromer CROM Glycoprotein (DAF or CD55,regulates 1 complement fractions C3 and C5, attached to the membrane byGPI). 022 Knops KN Glycoprotein (CR1 or CD35, immune complex 1receptor). 023 Indian IN Glycoprotein (CD44). 11 024 Ok OK Glycoprotein(CD147). 19 025 Raph MER2 Transmembrane glycoprotein. 11 026 JMH JMHProtein (fixed to cell membrane by GPI). 6 027 Ii I Branched(I)/unbranched (i) polysaccharide. 6 028 Globoside GLOB Glycolipid.Antigen P. 3 029 GIL GIL Aquaporin 3. 9 030 Rh-associated glycoproteinHemolytic Disease of the Newborn

The hemolytic condition occurs when there is an incompatibility betweenthe blood types of the mother and the fetus. Multiple differentantigen-antibody incompatibilities are implicated as causative, and manyof these are preventable or immediately treatable after birth.

The most common cause of severe hemolytic diseases of newborns is amaternal-fetal mismatch in an antigen of the Rh (Rhesus) blood groupsystem (including the Rh factor). The Rh blood group system currentlyconsists of 50 defined blood-group antigens, among which the 5 antigensD, C, c, E, and e are the most clinically relevant. For example, thedisorder in the fetus due to Rh D antigen incompatibility is known aserythroblastosis fetalis, and this can be prevented through inoculationof the mother with IgG anti-D (anti-RhD) antibodies that bind to, andlead to the destruction of, fetal Rh D positive red blood cells thathave passed from the fetal circulation to the maternal circulation.Therefore, in an Rh− negative mother it can prevent sensitization of thematernal immune system to Rh D antigens, which can cause rhesus diseasein the current or in subsequent pregnancies.

Hemolytic disease of the newborn (anti-Kell₁) is the second most commoncause of severe hemolytic diseases of newborns after Rh disease.Anti-Kell₁ is becoming relatively more important as prevention of Rhdisease is also becoming more effective. Hemolytic disease of thenewborn (anti-Kell₁) is caused by a mismatch between the Kell antigensof the mother and fetus. About 91% of the population are Kell₁ negativeand about 9% are Kell₁ positive. A fraction of a percentage arehomozygous for Kell₁. Therefore, about 4.5% of babies of a Kell₁negative mother are Kell₁ positive.

The disease results when maternal antibodies to Kell₁ are transferred tothe fetus across the placental barrier. These antibodies can causesevere anemia by interfering with the early proliferation of red bloodcells as well as causing alloimmune hemolysis. Very severe disease canoccur as early as 20 weeks gestation. Hydrops fetalis can also occurearly.

Determination of Fetal DNA Content in a Maternal Sample Using FetalAutosomal Polymorphisms and Genetic Variations

In each maternally-derived sample, the DNA from a fetus will haveapproximately 50% of its loci inherited from the mother and 50% of theloci inherited from the father. Determining the loci contributed to thefetus from paternal sources can allow the estimation of fetal DNA in amaternal sample, and thus provide information used to calculate thestatistically significant differences in chromosomal frequencies forchromosomes of interest.

In certain aspects, the determination of fetal polymorphisms requirestargeted SNP and/or mutation analysis to identify the presence of fetalDNA in a maternal sample. In some aspects, the use of prior genotypingof the father and mother can be performed. For example, the parents mayhave undergone such genotype determination for identification of diseasemarkers, e.g., determination of the genotype for disorders such ascystic fibrosis, muscular dystrophy, spinal muscular atrophy or even thestatus of the RhD gene may be determined. Such difference inpolymorphisms, copy number variants or mutations can be used todetermine the percentage fetal contribution in a maternal sample.

In one preferred aspect, the percent fetal cell free DNA in a maternalsample can be quantified using multiplexed SNP detection without usingprior knowledge of the maternal or paternal genotype. In this aspect,two or more selected polymorphic nucleic acid regions with a known SNPin each region are used. In a preferred aspect, the selected polymorphicnucleic acid regions are located on an autosomal chromosome that isunlikely to be aneuploidy, e.g. Chromosome 2. The selected polymorphicnucleic acid regions from the maternal are amplified. In a preferredaspect, the amplification is universal. In a preferred embodiment, theselected polymorphic nucleic acid regions are amplified in one reactionin one vessel. Each allele of the selected polymorphic nucleic acidregions in the maternal sample is determined and quantified. In apreferred aspect, high throughput sequencing is used for suchdetermination and quantification. Loci are identified where the maternaland fetal genotypes are different, e.g., the maternal genotype ishomozygous and the fetal genotype is heterozygous. This identificationis done by observing a high relative frequency of one allele (>80%) anda low relative frequency (<20% and >0.15%) of the other allele for aparticular selected nucleic acid region. The use of multiple loci isparticularly advantageous as it reduces the amount of variation in themeasurement of the abundance of the alleles. All or a subset of the locithat meet this requirement are used to determine fetal concentrationthrough statistical analysis. In one aspect, fetal concentration isdetermined by summing the low frequency alleles from two or more locitogether, dividing by the sum of the high frequency alleles andmultiplying by two. In another aspect, the percent fetal cell free DNAis determined by averaging the low frequency alleles from two or moreloci, dividing by the average of the high frequency alleles andmultiplying by two.

For many alleles, maternal and fetal sequences may be homozygous andidentical, and as this information is not distinguishing betweenmaternal and fetal DNA it is not useful in the determination of percentfetal DNA in a maternal sample. The present invention utilizes allelicinformation where there is a distinguishable difference between thefetal and maternal DNA (e.g., a fetal allele containing at least oneallele that differs from the maternal allele) in calculations of percentfetal. Data pertaining to allelic regions that are the same for thematernal and fetal DNA are thus not selected for analysis, or areremoved from the pertinent data prior to determination of percentagefetal DNA so as not to swamp out the useful data.

Exemplary methods for quantifying fetal DNA in maternal plasma can befound, e.g., in Chu et al., Prenat Diagn 2010; 30:1226-1229, which isincorporated herein by reference.

In one aspect, selected nucleic acid regions may be excluded if theamount or frequency of the region appears to be an outlier due toexperimental error, or from idiopathic genetic bias within a particularsample. In another aspect, selected nucleic acids may undergostatistical or mathematical adjustment such as normalization,standardization, clustering, or transformation prior to summation oraveraging. In another aspect, selected nucleic acids may undergo bothnormalization and data experimental error exclusion prior to summationor averaging.

In a preferred aspect, 12 or more loci are used for the analysis. Inanother preferred aspect, 24 or more loci are used for the analysis. Inanother preferred aspect, 48 or more loci are used for the analysis. Inanother aspect, one or more indices are used to identify the sample, thelocus, the allele or the identification of the nucleic acid.

In one preferred aspect, the percentage fetal contribution in a maternalsample can be quantified using tandem SNP detection in the maternal andfetal alleles. Techniques for identifying tandem SNPs in DNA extractedfrom a maternal sample are disclosed in Mitchell et al, U.S. Pat. No.7,799,531 and U.S. patent application Ser. Nos. 12/581,070, 12/581,083,12/689,924, and 12/850,588. These describe the differentiation of fetaland maternal loci through detection of at least one tandem singlenucleotide polymorphism (SNP) in a maternal sample that has a differenthaplotype between the fetal and maternal genome. Identification andquantification of these haplotypes can be performed directly on thematernal sample, as described in the Mitchell et al. disclosures, andused to determine the percent fetal contribution in the maternal sample.

Determination of Fetal DNA Content in a Maternal Sample Using EpigeneticAllelic Ratios

Certain genes have been identified as having epigenetic differencesbetween the placenta and maternal blood cells, and such genes arecandidate loci for fetal DNA markers in a maternal sample. See, e.g.,Chim SSC, et al. Proc Natl Acad Sci USA (2005); 102:14753-14758. Theseloci, which are unmethylated in the placenta but not in maternal bloodcells, can be readily detected in maternal plasma and were confirmed tobe fetus specific. Unmethylated fetal DNA can be amplified with highspecificity by use of methylation-specific PCR (MSP) even when suchfetal DNA molecules were present among an excess of background plasmaDNA of maternal origin. The comparison of methylated and unmethylatedamplification products in a maternal sample can be used to quantify thepercent fetal DNA contribution to the maternal sample by calculating theepigenetic allelic ratio for one or more of such sequences known to bedifferentially regulated by methylation in the fetal DNA as compared tomaternal DNA.

To determine methylation status of nucleic acids in a maternal sample,the nucleic acids of the sample are subjected to bisulfate conversion ofthe samples and then subjected them to MSP, followed by allele-specificprimer extension. Conventional methods for such bisulphite conversioninclude, but are not limited to, use of commercially available kits suchas the Methylamp™ DNA Modification Kit (Epigentek, Brooklyn, N.Y.).Allelic frequencies and ratios can be directly calculated and exportedfrom the data to determine the relative percentage of fetal DNA in thematernal sample.

Use of Percent Fetal Cell Free DNA to Detect Aneuploidy

Once the percent fetal cell free DNA has been calculated, this data maybe combined with methods for aneuploidy detection to determine thelikelihood that a maternal sample may contain an aneuploidy. In oneaspect, an aneuploidy detection methods that utilizes analysis of randomDNA segments is used, such as that described in, e.g., Quake, U.S.patent application Ser. No. 11/701,686; Shoemaker et al., U.S. patentapplication Ser. No. 12/230,628. In a preferred aspect, aneuploidydetection methods that utilize analysis of selected nucleic acid regionsis used. In this aspect, the percent fetal cell free DNA for a sample iscalculated. The chromosomal ratio for that sample, a chromosomal ratiofor the normal population and a variation for the chromosomal ratio forthe normal population is determined, as described herein. In onepreferred aspect, the chromosomal ratio and its variation for the normalpopulation are determined from normal samples that have a similarpercentage of fetal DNA. An expected aneuploidy chromosomal ratio for aDNA sample with that percent fetal cell free DNA is calculated by addingthe percent contribution from the aneuploidy chromosome. The chromosomalratio for the sample may then be compared to the chromosomal ratio forthe normal population and to the expected aneuploidy chromosomal ratioto determine statistically, using the variation of the chromosomalratio, to determine if the sample is more likely normal or aneuploidy,and the statistical probability that it is one or the other.

In a preferred aspect, the selected regions of a maternal sample includeboth regions for determination of fetal DNA content as well asnon-polymorphic regions from two or more chromosomes to detect a fetalchromosomal abnormality in a single reaction. The single reaction helpsto minimize the risk of contamination or bias that may be introducedduring various steps in the assay system which may otherwise skewresults when utilizing fetal DNA content to help determine the presenceor absence of a chromosomal abnormality.

In other aspects, a selected region or regions may be utilized both fordetermination of fetal DNA content as well as detection of fetalchromosomal abnormalities. The alleles for selected regions can be usedto determine fetal DNA content and these same selected regions can thenbe used to detect fetal chromosomal abnormalities ignoring the allelicinformation. Utilizing the same regions for both fetal DNA content anddetection of chromosomal abnormalities may further help minimize anybias due to experimental error or contamination.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees centigrade, and pressure is at or nearatmospheric.

Example 1: General Aspects of the Assay Systems of the Invention

A number of assay formats were tested to demonstrate the ability toperform selective amplification and detection of independent loci todemonstrate multiplexed, ligation-based detection of a large number(e.g., 96 or more) of loci of interest. These loci included loci thatwere indicative of the presence of a particular chromosome or thepresence or absence of a mutation or polymorphism in a particularallele.

These assays were designed based on human genomic sequences, and eachinterrogation consisted of two fixed sequence oligos per selected locusinterrogated in the assay. The first oligo, complementary to the 3′region of a genomic region, comprised the following sequential (5′ to3′) oligo elements: a universal PCR priming sequence common to allassays: TACACCGGCGTTATGCGTCGAGAC (SEQ ID NO:1); a nine nucleotideidentification index specific to the selected locus; a 9 base locus- orlocus/allele-specific sequence that acts as a locus index in the firstSNP-independent set and a locus/allele index in thepolymorphism-specific second set; a hybridization breaking nucleotidewhich is different from the corresponding base in the genomic locus; anda 20-24 bp sequence complementary to the selected genomic locus. Incases where a SNP or mutation was detected in this portion of theselected genomic locus, the allele-specific interrogation set consistedof two first fixed sequence tandem ligation primers, each with adifferent locus/allele index and a different allele-specific base at theSNP position. These first oligos were designed for each selected nucleicacid to provide a predicted uniform T_(m) with a two degree variationacross all interrogations in the assay set.

The second fixed sequence oligo, complementary to the 5′ region of thegenomic loci, comprised the following sequential (5′ to 3′) elements: a20-24b sequence complimentary to the 5′ region in the genomic locus; ahybridization breaking nucleotide different from the corresponding basein the genomic locus; and a universal PCR priming sequence which wascommon to all third oligos in the assay set: ATTGCGGGGACCGATGATCGCGTC(SEQ ID NO:2).

In cases where a SNP or mutation was detected in the selected genomiclocus, the allele-specific interrogation set consisted of two tandemligation primers, each with a different locus/allele index and adifferent allele-specific base at the mutation/SNP position. This secondfixed sequence oligo was designed for each selected nucleic acid toprovide a predicted uniform T_(m) with a two degree variation across allinterrogations in the assay set that was substantially the same T_(m)range as the first oligo set.

In certain tested aspects, one or more bridging oligos were used thatwere complementary to the genomic locus sequence between the regioncomplementary to the first and second fixed sequence oligos used foreach selected locus. In specific aspects tested, more than one bridgingoligo was used to span the gap between the fixed sequenceoligonucleotides, and the one or more bridging oligo may optionally bedesigned to identify one or more mutations or SNPs in the sequence. Thelength of the bridging oligonucleotides used in the assay systems variedfrom 5 to 36 base pairs.

All oligonucleotides used in the tandem ligation formats weresynthesized using conventional solid-phase chemistry. The second fixedsequence oligos and the bridging oligonucleotides were synthesized with5′ phosphate moieties to enable ligation to 3′ hydroxyl termini ofadjacent oligonucleotides.

Example 2: Preparation of DNA for Use in Tandem Ligation Procedures

Genomic DNA from a Caucasian male (NA12801) or a Caucasian female(NA11995) was obtained from Coriell Cell Repositories (Camden, N.J.) andfragmented by acoustic shearing (Covaris, Woburn, Mass.) to a meanfragment size of approximately 200 bp.

The Coriell DNA was biotinylated using standard procedures. Briefly, theCovaris fragmented DNA was end-repaired by generating the followingreaction in a 1.5 ml microtube: 5 μg DNA, 12 μl 10× T4 ligase buffer(Enzymatics, Beverly Mass.), 50 U T4 polynucleotide kinase (Enzymatics,Beverly Mass.), and H₂0 to 120 μl. This was incubated at 37° C. for 30minutes. The DNA was diluted using 10 mM Tris 1 mM EDTA pH 8.5 todesired final concentration of ˜2 ng/μl.

5 μl DNA was placed in each well of a 96-well plate, and the platesealed with an adhesive plate sealer and spun for 10 seconds at 250×g.The plate was then incubated at 95° C. for 3 minutes, cooled to 25° C.,and spun again for 10 seconds at 250×g. A biotinylation master mix wasprepared in a 1.5 ml microtube to final concentration of: 1×TdT buffer(Enzymatics, Beverly, Mass.), 8U TdT (Enzymatics, Beverly, Mass.), 250μM CoCl₂, 0.01 nmol/μl biotin-16-dUTP (Roche, Nutley N.J.), and H₂0 to1.5 ml. 15 μl of the master mix was aliquoted into each well of a 96well plate, and the plate sealed with adhesive plate sealer. The platewas spun for 10 seconds at 250×g and incubated for 37° C. for 60minutes. Following incubation, the plate was spun again for 10 secondsat 250×g, and 7.5 μl precipitation mix (1 μg/μl Dextran Blue, 3 mMNaOAC) was added to each well.

The plate was sealed with an adhesive plate sealer and mixed using anIKA plate vortexer for 2 minutes at 3000 rpm. 27.5 μl of isopropanol wasadded into each well, the plate sealed with adhesive plate sealer, andvortexed for 5 minutes at 3000 rpm. The plate was spun for 20 minutes at3000×g, the supernatant was decanted, and the plate inverted andcentrifuged at 10×g for 1 minute onto an absorbent wipe. The plate wasair-dried for 5 minutes, and the pellet resuspended in 30 μl 10 mM TrispH8.0, 1 mM EDTA.

Example 3: Exemplary Assay Formats Using Tandem Ligation

Numerous tandem ligation assay formats using the biotinylated DNA weretested to illustrate proof of concept for the assay systems of theinvention, and demonstrated the ability to perform highly multiplexed,targeted detection of a large number of independent loci using theseries of different assay formats. The exemplary assay systems of theinvention were designed to comprise 96 or more interrogations per lociin a genetic sample, and in cases where SNPs were detected the assayformats utilized 192 or more separate interrogations, each utilizing thedetection of different alleles per 96 loci in genetic samples. Theexamples described for each assay format utilized two different sets offixed sequence oligonucleotides and/or bridging oligos (as described inExample 1), comprising a total 96 or 192 interrogation reactions for theselected loci depending upon whether or not SNPs were identified.

A first exemplary assay format used locus-specific fixed sequence oligosand bridging oligos, where there was a one base gap between the firstfixed sequence oligo and the bridging oligos, and a second one base gapbetween the bridging oligos and the second fixed sequence oligo. Each ofthe two gaps encompassed two different SNPs. In this format, a DNApolymerase was used to incorporate each of the SNP bases, and ligase wasused to seal the nicks formed thereby. SNP base discrimination derivedfrom the fidelity of base incorporation by the polymerase, and in theevent of mis-incorporation, the tendency of ligase to not seal nicksadjacent to mismatched bases.

The second exemplary assay format used two locus-specific fixed sequenceoligonucleotides without a bridging oligo, where there was a ˜15-35 basegap between the fixed sequence oligos, and where the gap spanned one ormore SNPs. In this format, a polymerase was used to incorporate themissing bases of the gap, and a ligase was used to seal the nick formedthereby. SNP base discrimination derived from the fidelity of baseincorporation by the polymerase, and in the event of misincorporation,the tendency of ligase to not seal nicks adjacent to mismatched bases.

A third exemplary assay format used allele-specific first and secondfixed sequence oligos without a bridging oligo, where there was a ˜15-35base gap between the first and second fixed sequence oligos, and wherethe gap spanned one or more SNPs. Two separate allele-specific firstfixed sequence oligos and two separate allele-specific second fixedsequence oligos were used. A polymerase was used to incorporate themissing bases, and a ligase was used to seal the nick formed thereby.SNP base discrimination derived from hybridization specificity, thetendency of non-proofreading polymerase to not extend annealed primerswith mismatches near the 3′ end, and the tendency of the ligase to notseal nicks adjacent to mismatched bases.

A fourth exemplary format used allele-specific fixed sequence oligos anda locus-specific bridging oligo. In this format, two separate fixedsequence oligos complementary to the 3′ end of the loci of interest, thefirst with a 3′ base specific for one allele of the targeted SNP, andthe second with a 3′ base specific for the other allele of the targetedSNP. Similarly, two separate second fixed sequence oligos were used, thefirst with a 5′ base specific for one allele of a second targeted SNP,and the second with a 5′ base specific for the other allele of thesecond targeted SNP. The bridging oligos were complementary to theregion directly adjacent to the locus regions complementary to the firstand second fixed sequence oligos, and thus no polymerase was neededprior to ligation. Ligase was used to seal the nicks between the fixedsequence oligos and the bridging oligo. SNP base discrimination in thisassay format derived from hybridization specificity and the tendency ofthe ligase to not seal nicks adjacent to mismatched bases. Thisexemplary format was tested using either T4 ligase or Taq ligase forcreation of the contiguous template, and both were proved effective inthe reaction as described below.

A fifth exemplary format used locus-specific fixed sequence oligos thatwere complementary to adjacent regions on the nucleic acid of interest,and thus no gap was created by hybridization of these oligos. In thisformat, no polymerase was required, and a ligase was used to seal thesingle nick between the oligos.

A sixth exemplary format used allele-specific fixed sequence oligos andlocus-specific bridging oligos, where there was a short base gap of fivebases between the loci region complementary to the fixed sequenceoligos. The locus-specific bridging oligo in this example was a 5 mercomplementary to the regions directly adjacent to the regionscomplementary to the first and second fixed sequence oligos. In thisformat, no polymerase was required, and a ligase was used to seal thetwo nicks between the oligos.

A seventh exemplary format used locus-specific fixed sequence oligos anda locus-specific bridging oligo, where there was a shorter base gap offive bases containing a SNP in the region complementary to the bridgingoligo. Allele-specific bridging oligos corresponding to the possibleSNPs were included in the hybridization and ligation reaction. In thisformat, no polymerase was required, and a ligase was used to seal thetwo nicks between the oligos. SNP base discrimination in this assayformat derived from hybridization specificity and the tendency of theligase to not seal nicks adjacent to mismatched bases.

An eighth exemplary format used locus-specific fixed sequence oligos andtwo adjacent locus-specific bridging oligos, where there was a 10 basegap between the regions complementary to the first and second fixedsequence oligos. Locus-specific bridging oligos were included in theligation reaction, with the gap requiring two contiguous 5 mers tobridge the gap. In this format, no polymerase was required, and a ligasewas used to seal the three nicks between the oligos.

For each of the above-described assay formats, an equimolar pool (40 nMeach) of sets of first and second loci- or allele-specific fixedsequence oligonucleotides was created from the oligos prepared as setforth in Example 2. A separate equimolar pool (20 μM each) of bridgingoligonucleotides was likewise created for the assay processes based onthe sequences of the selected genomic loci.

100 μs of strepavidin beads were transferred into the wells of a 96 wellplate, and the supernatant was removed. 60 μl BB2 buffer (100 mM Tris pH8.0, 10 mM EDTA, 500 mM NaCl₂, 58% formamide, 0.17% TWEEN™-80), 10 μL 40nM fixed sequence oligo pool and 30 μL of the biotinylated template DNAprepared in Example 2 were added to the beads. The plate was sealed withan adhesive plate sealer and vortexed at 3000 rpm until beads wereresuspended. The oligos were annealed to the template DNA by incubationat 70° C. for 5 minutes, followed by slow cooling to room temperature.

The plate was placed on a raised bar magnetic plate for 2 minutes topull the magnetic beads and associated DNA to the side of the wells. Thesupernatant was removed by pipetting, and was replaced with 50 μl of 60%BB2 (v/v in water). The beads were resuspended by vortexing, placed onthe magnet again, and the supernatant was removed. This bead washprocedure was repeated once using 50 μl 60% BB2, and repeated twice moreusing 50 μl wash buffer (10 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaCl₂).

The beads were resuspended in 37 μl ligation reaction mix consisting of1×Taq ligase buffer (Enzymatics, Beverly, Mass.), 1U Taq ligase, and 2μM bridging oligo pool (depending on the assay format), and incubated at37° C. for one hour. Where appropriate, and depending on the assayformat, a non-proofreading thermostable polymerase plus 200 nM each dNTPwas included in this mixture. The plate was placed on a raised barmagnetic plate for 2 minutes to pull the magnetic beads and associatedDNA to the side of the wells. The supernatant was removed by pipetting,and was replaced with 50 μL wash buffer. The beads were resuspended byvortexing, placed on the magnet again, and the supernatant was removed.The wash procedure was repeated once.

To elute the products from the strepavidin beads, 30 μl of 10 mM Tris 1mM EDTA, pH 8.0 was added to each well of 96-well plate. The plate wassealed and mixed using an IKA vortexer for 2 minutes at 3000 rpm toresuspend the beads. The plate was incubated at 95° C. for 1 minute, andthe supernatant aspirated using an 8-channel pipetter. 25 μl ofsupernatant from each well was transferred into a fresh 96-well platefor universal amplification.

Example 4: Universal Amplification of Tandem Ligated Products

The polymerized and/or ligated nucleic acids were amplified usinguniversal PCR primers complementary to the universal sequences presentin the first and second fixed sequence oligos hybridized to the loci ofinterest. 25 μl of each of the reaction mixtures of Example 3 were usedin each amplification reaction. A 50 μl universal PCR reactionconsisting of 25 μl eluted ligation product plus 1× Pfusion buffer, 1MBetaine, 400 nM each dNTP, 1 U Pfusion error-correcting thermostable DNApolymerase (Thermo Fisher, Waltham Mass.), and the following primerpairs: TAATGATACGGCGACCACCGAGATCTACACCGGCGTTATGCGTCGAGA (SEQ ID NO:3)and TCAAGCAGAAGACGGCATACGAGATXAAACGACGCGATCATCGGTCCCCGCAA (SEQ ID NO:4),where X represents one of 96 different sample indices used to uniquelyidentify individual samples prior to pooling and sequencing. The PCR wascarried out under stringent conditions using a BioRad Tetrad™thermocycler.

10 μl of universal PCR product from each of the samples were pooled andthe pooled PCR product was purified using AMPureXP™ SPRI beads(Beckman-Coulter, Danvers, Mass.), and quantified using Quant-iT™PicoGreen, (Invitrogen, Carlsbad, Calif.).

Example 5: Detection and Analysis of Selected Loci

The purified PCR products of each assay format were sequenced on asingle lane of a slide on an Illumina HISEQ™ 2000 (Illumina, San Diego,Calif.). Sequencing runs typically give rise to ˜100M raw reads, ofwhich ˜85M (85%) mapped to expected assay structures. This translated toan average of ˜885K reads/sample across the experiment, and (in the caseof an experiment using 96 loci) 9.2K reads/replicate/locus across 96loci. The mapped reads were parsed into replicate/locus/allele counts,and various metrics were computed for each condition, including:

Yield: a metric of the proportion of input DNA that was queried insequencing, computed as the average number of unique reads per locus(only counting unique identification index reads per replicate/locus)divided by the total number of genomic equivalents contained in theinput DNA.

80 percentile locus frequency range: a metric of the locus frequencyvariability in the sequencing data, interpreted as the fold range thatencompasses 80% of the loci. It was computed on the distribution oftotal reads per locus, across all loci, as the 90^(th) percentile oftotal reads per locus divided by the 10^(th) percentile of the totalreads per locus.

SNP error rate: a metric of the error rate at the SNP position, andcomputed as the proportion of reads containing a discordant base at theSNP position.

These results are summarized in Table 1:

TABLE 1 Results Summary of Tandem Ligation Assay Formats FIXED 80%SEQUENCE BRIDGING LOC SNP ASSAY OLIGO (1^(st) and/or OLIGO ENZYME FREQERROR FORMAT 2^(nd)) USED USED YIELD RANGE RATE 1 LOCUS-SPECIFIC Locuspol + lig 9.5% 5.3 0.18% specific 2 LOCUS-SPECIFIC No pol + lig 1.4%58.3 0.19% 3 ALLELE- No pol + lig 0.4% 61.7 1.00% SPECIFIC 4 ALLELE-Locus Taq lig 5.0% 5.9 0.92% SPECIFIC specific 4 ALLELE- Locus T4 lig5.3% 4.4 0.95% SPECIFIC specific 5 LOCUS-SPECIFIC No Taq lig 22.5% 1.7N/A 6 LOCUS-SPECIFIC Locus Taq lig 12.5 2.9 N/A specific 7LOCUS-SPECIFIC Allele Taq lig 14.3 2.8 0.20% specific 8 LOCUS-SPECIFIC 2Locus Taq lig 18.5% 2.8 N/A specific

Table 1 indicates that the locus-specific tandem ligation assay using abridging oligo converted template DNA into targeted product with highyield (˜10%), with a high proportion of product derived from targetedloci (15% of reads did not contain expected assay structures), withlimited locus bias (80% of loci fall within a ˜5-fold concentrationrange), and with high SNP accuracy (0.2% SNP error rate). Thelocus-specific tandem ligation assay without the use of a bridging oligoproduced reduced yields and substantial locus bias, but still producedhigh accuracy SNP genotyping data. The allele-specific tandem ligationassay with a bridging oligo produced intermediate yields compared to thelocus-specific assay using both T4 and Taq ligase, but still producedlimited locus bias and high accuracy SNP genotyping data. Theallele-specific tandem ligation assay without a bridging producedreduced yields and substantial locus bias, but still produced highaccuracy SNP genotyping data.

Assay formats six through eight showed that template DNA can beconverted into targeted product with high yield (12-18%), with a highproportion of product derived from targeted loci (˜76% of readscontained expected assay structures), and with limited locus bias (80%of loci fall within a 2-3-fold concentration range). FIG. 5 illustratesthe genotyping performance that was obtained using assay format seven,comparing the sequence counts for the two alleles of all polymorphicassays observed in a single sample. Note the clear separation of thehomozygous and heterozygous clusters, as well as the low backgroundcounts observed amongst the homozygous clusters.

Example 6: Detection of Aneuploidy in Patient Samples from PregnantSubjects

The assay systems of the invention were used in the detection ofpolymorphisms and chromosomal abnormalities in two separate cohorts ofpregnant females. A first cohort of 190 normal, 36 T21, and 8 T18pregnancies and a second cohort of 126 normal, 36 T21, and 8 T18pregnancies were tested for fetal aneuploidy. The chromosomalaneuploidies were detected using 576 chromosome 21 and 576 chromosome 18assays, pooled together and assayed in a single reaction, as set forthbelow.

The elements used in the aneuploidy detection assays are illustrated inFIG. 6. The cfDNA 601 isolated from maternal samples was used as atemplate for hybridization, ligation, and amplification of multipleselected loci from both chromosome 21 and chromosome 18 in each maternalsample. Three oligonucleotides were hybridized to each selected locus tocreate ligation products for amplification and detection. The left (orfirst) fixed sequence oligonucleotide comprised a region complementaryto a selected locus 609 and a first universal primer region 611. Theright (or second) fixed sequence oligonucleotide 605 comprised a secondregion complementary to the selected locus 613 and a second universalprimer region 615. The bridging oligonucleotides 607 used were designedso that each would hybridize to bridging regions of two or more selectedloci used in the aneuploidy detection assay. When the fixed sequenceoligonucleotides 603, 605 and the bridging oligonucleotide 607hybridized to the complementary region on the cfDNA 601, their terminiformed two nicks. Upon ligation of the hybridized oligonucleotides tothe cfDNA, a ligation product was created for each selected locuscomprising 603, 605 and 607 which was used as a template foramplification primers 619, 621.

Two amplification primers 619, 621 comprising regions complementary tothe first and second universal primer regions, respectively, were thenused to amplify the ligation product. This amplification productcomprised the sequence of the selected locus. The right amplificationprimer also comprised a sample index 617 to identify the particularsample from which the locus was obtained in the multiplexed assay.Amplification with 96 distinct right amplification primers 629 enabledpooling and simultaneous sequencing of 96 different amplificationproducts on a single lane.

The amplification primers 619, 621 also contained a left clustersequence 623 (TAATGATACGGCGACCACCGA)(SEQ ID NO:7) and a right clustersequence 625 (ATCTCGTATGCCGTCTTCTGCTTGA)(SEQ ID NO:8) that supportedcluster amplification for sequencing using the Illumina HISEQ™ 2000system (Illumina, San Diego, Calif.). A sequencing primer 627 comprisingthe first universal primer sequence was used to determine the sequenceof the amplification product, and a second sequencing primer 629 wasused to determine the sample index 617 of the amplification product.

Briefly, approximately 10 mL peripheral blood was collected from eachpatient into a BCT tube (Streck, Omaha, Nebr.), which was shipped viaovernight courier to Tandem Diagnostics. Plasma was isolated from BCTtubes within 72 h of blood collection by centrifugation at 1600 g for 10m. The plasma was transferred to a second tube and centrifuged at 16000g for 10 m to remove any remaining cells. cfDNA was isolated from 4-5 mLplasma per patient. Approximately 15 ng cfDNA was isolated from eachpatient sample and arrayed into individual wells of a 96 well plate. Allsubsequent processing occurred on multiplexed batches of up to 96 cfDNApatient samples per array system method.

cfDNA isolated from the maternal samples in each well was biotinylatedprecipitated and resuspended in 30 uL TE as in Example 3 above. Thebiotinylated template DNA was mixed with 100 ug MyOneC1streptavidin-coated magnetic beads (Life Technologies, Carlsbad,Calif.), 60 μl BB2 buffer (100 mM Tris pH 8.0, 10 mM EDTA, 500 mM NaCl₂,58% formamide, 0.17% TWEEN™-80), and 10 μL of pooled 40 nM left 603 andright 605 fixed sequence oligonucleotides. The mixture was heated to 70°C., and cooled 2 hours. The beads were then magnetically immobilized tothe side of the well, washed twice with 50 uL 60% BB2 (v/v with H2O),washed twice more with 50 μl wash buffer (10 mM Tris pH 8.0, 1 mM EDTA,50 mM NaCl2), and then resuspended in a 50 μL reaction containing 1U Taqligase (Enzymatics, Beverly Mass.), 1×Taq ligase buffer (Enzymatics),and 10 uM of a 5′-phosphorylated 5 mer bridging oligonucleotide 607. Themixture was incubated at 37° C. for 1 hour. The beads were againmagnetically immobilized to the side of the well, washed twice with 50uL wash buffer and then resuspended in 30 μL TE.

The ligation products were eluted from the immobilized beads byincubation at 95° C. for 3 minutes. The eluted ligation products wereamplified by 26 cycles of PCR in a 50 uL reaction containing 1U Pfusion(Finnzymes), 1M Betaine, 1× Pfusion buffer, and 400 nM left and rightamplification primers (619, 621 respectively). The right primercontained a 7 base sample index (617) that enabled 96 sample multiplexedsequencing on the HISEQ2000 (Illumina, San Diego, Calif.). The sequenceof the left fixed sequence oligo was:

(SEQ ID NO: 5) TAATGATACGGCGACCACCGAGATCTACACCGGCGTTATGCGTCGAGAC

And the sequence of the right fixed sequence oligo was:

(SEQ ID NO: 6) TCAAGCAGAAGACGGCATACGAGATNNNNNNNAAACGACGCGATCATCGGTCCCCGCAAT

Amplification products from a single 96 well plate were pooled in equalvolume, and the pooled amplification products were purified withAMPureXP™ SPRI beads (Beckman-Coulter, Danvers, Mass.) according to themanufacturer's instructions. Each purified pooled library was used astemplate for cluster amplification on an Illumina TruSeq v2 SR clusterkit flow cell (Illumina, San Diego, Calif.) according to manufacturer'sprotocols. The slide was processed on an Illumina HISEQ™ 2000 (Illumina,San Diego, Calif.) to produce 56 bases of locus-specific sequence from aleft sequence primer 623 and a separate read of 8 bases of samplespecific sequence was obtained from the second sequence primer 625. Anaverage of 903K raw reads per sample were collected. An average of 876K(97%) of the reads was assigned to expected assay structures.

FIG. 7 shows exemplary data for a subset of the patient samples from thesecond cohort, which were all analyzed in one multiplexed assay on asingle lane of a sequencing run. Initially 96 different samples were runin this particular run, but six samples were later excluded from thisanalytical set as not meeting sample quality control thresholds.

A trimmed mean was calculated for each chromosome 18 and chromosome 21for the samples based on reads produced in the assay. The trimmed meanwas computed by removing 10% of high and low counts for each chromosomeby sample. The detected amplification products corresponding to thevarious selected loci were used to compute a chromosome 21 proportionmetric and a chromosome 18 proportion metric for each sample. Forchromosome 21 proportion, this was calculated as the trimmed mean ofcounts in the 384 chromosome 21 selected loci divided by the sum oftrimmed means of counts for all 576 chromosome 21 loci and 576chromosome 18 loci for each sample.

On average 834 read counts were observed per selected locus in thematernal samples of the first cohort, and 664 read counts were observedper selected locus from the second cohort. These counts were used tocompute chromosome proportion z-scores for chromosome 21 and chromosome18.

Briefly, the z-scores were calculated by scaling the median per locuscount to a common value (e.g., 1000) for each sample, and the scaledcounts were transformed by log base 2. An RMA log linear modeling andmedian polish were performed (Bolstad, B. M et al. (2003) Bioinformatics19(2):185-193; Rafael. A. (2003) Nucleic Acids Research 31(4):e15;Irizarry, R A et al. (2003) Biostatistics 4(2):249-64) to estimatechromosome effects, locus effects, sample effects, and residuals. Theestimated chromosome effects were set to a common value, e.g., 0, and2^(chromosome effect+sample effect+residual) was calculated for eachlocus to create normalized counts. The Z scores were scaled usingiterative censoring so that they had a mean of 0 and a standarddeviation of 1.

Data obtained from the first cohort of samples was used to determinefirst cohort z-scores for chromosome 21 and chromosome 18 areillustrated in FIGS. 8 and 9, respectively. The normal samples are shownas dark grey diamonds, and the samples with a trisomy are shown as lightgrey diamonds. 179/180 (99.4%) normal samples (dark grey diamonds) hadz-scores <3; one normal sample had a chromosome 21 z-score of 3.4 and achromosome 18 z-score of 3.0. 35/35 (100%) T21 and 7/7 (100%) T18samples had chromosome proportion z-scores >3. The mean T18 z-score was8.5, and the range was 5.8-10.9. The mean T21 z-score was 11.5, and therange was 6.1-19.8.

The data provided in FIG. 7 was combined with data from the remainingsamples of the second cohort to determine z-scores for chromosome 21 andchromosome 18 are illustrated in FIGS. 10 and 11, respectively. Thenormal samples are shown as dark grey diamonds, and the samples with atrisomy are shown as light grey diamonds. 125/125 normal samples hadz-scores <3, 36/36 (100%) T21 and 8/8 (100%) T18 samples hadz-scores >3. The mean T18 z-score was 9.5 and the range was 5.1-19.8.The mean T21 z-score was 11.4 and the range was 3.4-21.8.

In addition to the detection of aneuploidy in these cohorts, specificpolymorphisms were also determined for these samples in a same assay.Specific information was obtained for individual loci as well as moregeneral polymorphic information, such as the number of loci in which thefetal locus displayed a single nucleotide polymorphism in one alleledifferent from the single nucleotide polymorphisms at the maternal locus(FIG. 7, #Locus DiffPoly). This determination also identified thepresence of specific polymorphisms in the fetal genome. For example, thestatus of three exemplary polymorphism were determined using acombination of bridging oligos that were designed to bind to both the Aand the T residue in the following exemplary polymorphic regions:

TABLE 2 Individual Polymorphisms Queried Using the Invention AssayChromosome               Location         RSIDCh01                     01_010303942     rs11582123 (SEQ ID NO: 7)TTTACATGTCTTTGGGCATTTTAGGT[A/T]GAGTGAAATCTAGGCCTTGCAAATCCh03                     03_098690592     rs2470750 (SEQ ID NO: 8)TTGTGTAACGTTAACCTCAGGGACCA[A/T]GAGATGTACTTAGTATTAATTTGCCCh04                     04_055495793     rs6815910 (SEQ ID NO: 9)GGAAGAAGTGCAGTGTAGTAGACAAC[A/T]CTGGCATTGTGTTTTGTGAACTGGG

TABLE 3 Predicted Maternal and Fetal Status for SNP rs11582123.Predicted Predicted Fetal Maternal Sample SNP A counts T counts StatusStatus 1 rs11582123 294 26 A/T A/A 2 181 134 A/A A/T 4 34 330 A/T T/T 5241 21 A/T A/A 6 166 134 A/T A/T 7 137 182 T/T A/T 8 199 135 A/A A/T 9 0267 T/T T/T 10 0 284 T/T T/T 11 151 154 A/T A/T 12 294 1 A/T A/A 13 131114 A/A A/T 14 118 159 T/T A/T 15 257 10 A/T A/A 16 309 31 A/T A/A 17 20289 A/T T/A 18 137 166 T/T A/T 19 138 143 A/T A/T 20 24 242 A/T T/T 21140 161 A/T A/T 22 159 118 A/A A/T 23 119 122 A/T A/T 24 0 250 T/T T/T25 0 285 T/T T/T 26 120 130 A/T A/T 28 134 113 A/A A/T 29 109 118 A/TA/T 30 0 271 T/T T/T 31 148 139 A/T A/T 32 29 253 A/T T/T 33 0 304 T/TT/T 34 0 278 T/T T/T 35 103 188 T/T A/T 36 18 269 A/T T/T 37 279 34 A/TA/A 38 0 250 T/T T/T 39 0 263 T/T T/T 40 136 142 A/T A/T 41 147 145 A/TA/T 42 15 270 A/T T/T 43 44 222 A/T T/T 44 140 159 T/T A/T 45 0 259 T/TT/T 46 1 304 T/T T/T 47 162 127 A/A A/T 48 0 335 T/T T/T 49 1 247 T/TT/T 50 153 154 A/T A/T 51 118 182 T/T A/T 52 145 134 A/T A/T 53 146 132A/T A/T 54 7 319 A/T T/T 55 152 174 T/T A/T 56 1 319 T/T T/T 57 147 150A/T A/T 58 136 157 T/T A/T 59 83 162 A/A T/T 60 14 215 A/T T/T 61 157121 A/A A/T 62 281 0 A/A A/A 63 0 260 T/T T/T 64 0 305 T/T T/T 65 18 252A/T T/T 66 0 303 T/T T/T 67 99 161 T/T A/T 68 141 127 A/T A/T 69 0 237T/T T/T 70 0 315 T/T T/T 71 132 139 A/T A/T 73 112 120 A/T A/T 75 1 268T/T T/T 76 166 123 A/A A/T 78 0 245 T/T T/T 79 12 264 A/T T/T 80 15 281A/T T/T 81 21 269 A/T T/T 82 108 160 T/T A/T 83 106 144 T/T A/T 84 137135 A/T A/T 85 115 151 T/T A/T 86 0 262 T/T T/T 87 0 269 T/T T/T 89 0284 T/T T/T 90 0 261 T/T T/T 91 143 137 A/T A/T 92 0 308 T/T T/T 93 1256 T/T T/T 94 158 105 A/A A/T 95 149 103 A/A A/T

TABLE 4 Predicted Maternal and Fetal Status for SNP rs2470750. PredictedPredicted Fetal Maternal Sample SNP A counts T counts Status Status 1rs2470750 243 15 A/T A/A 2 265 0 A/A A/A 4 170 107 A/A A/T 5 196 30 A/TA/A 6 141 144 A/T A/T 7 139 137 A/T A/T 8 272 0 A/A A/A 9 218 0 A/A A/A10 216 0 A/A A/A 11 228 0 A/A A/A 12 6 224 A/T T/T 13 126 93 A/A A/T 14125 123 A/T A/T 15 234 20 A/T A/A 16 147 113 A/A A/T 17 235 2 A/T A/A 18129 142 A/T A/T 19 132 114 A/A A/T 20 214 0 A/A A/A 21 1 245 T/T T/T 22141 111 A/A A/T 23 135 128 A/A A/T 24 121 160 T/T A/T 25 209 21 A/T A/A26 0 239 T/T T/T 27 203 4 A/T A/A 28 101 115 T/T A/T 29 212 10 A/T A/A30 86 101 T/T A/T 31 118 116 A/T A/T 32 135 121 A/A A/T 33 111 128 T/TA/T 34 120 118 A/T A/T 35 246 0 A/A A/A 36 113 115 A/T A/T 37 96 126 T/TA/T 38 107 88 A/A A/T 39 241 0 A/A A/A 40 116 118 A/T A/T 41 135 89 A/AA/T 42 129 85 A/A A/T 43 0 205 T/T T/T 44 138 88 A/A A/T 45 129 86 A/AA/T 46 108 123 T/T A/T 47 14 246 A/T T/T 48 129 148 T/T A/T 49 108 110A/T A/T 50 120 124 A/T A/T 51 212 22 A/T A/T 52 237 0 A/A A/A 53 104 147T/T A/T 54 134 126 A/T A/T 55 128 82 A/A A/T 56 225 5 A/T A/A 57 213 11A/T A/A 58 125 116 A/T A/T 59 226 1 A/A A/A 60 103 119 T/T A/T 61 84 91T/T A/T 62 130 104 A/A A/T 63 251 0 A/A A/A 64 243 0 A/A A/A 65 127 115A/A A/T 66 113 104 A/A A/T 67 26 190 A/T T/T 68 80 83 A/T A/T 69 122 132T/T A/T 70 0 235 T/T T/T 71 90 123 T/T A/T 73 174 0 A/A A/A 75 0 233 T/TT/T 76 220 0 A/A A/A 78 115 115 A/T A/T 79 112 144 T/T A/T 80 10 248 A/TT/T 81 241 0 A/A A/A 82 228 0 A/A A/A 83 243 16 A/T A/A 84 133 104 A/AA/T 85 101 99 A/T A/T 86 1 209 A/A A/A 87 224 7 A/T A/A 89 122 101 A/AA/T 90 130 89 A/A A/T 91 128 151 T/T A/T 92 231 0 A/A A/A 93 107 118 T/TA/T 94 93 100 A/T A/T 95 132 119 A/A A/T

TABLE 5 Predicted Maternal and Fetal Status for SNP rs6815910. PredictedPredicted Fetal Maternal Sample SNP A counts T counts Status Status 1rs6815910 295 32 A/T A/A 10 133 107 A/A A/T 11 115 131 T/T A/T 12 311 10A/T A/A 13 18 252 A/T T/T 14 132 178 T/T A/T 15 288 0 A/A A/A 16 325 1A/A A/A 17 11 276 A/T T/T 18 282 0 A/A A/A 19 131 133 A/T A/T 2 7 311A/T T/T 20 135 116 A/A A/T 21 121 140 T/T A/T 22 287 11 A/T A/A 23 148146 A/T A/T 24 185 138 A/A A/T 25 116 126 T/T A/T 26 235 0 A/A A/A 27288 0 A/A A/A 28 242 0 A/A A/A 29 239 12 A/T A/A 30 235 24 A/T A/A 31126 148 T/T A/T 32 25 256 A/T T/T 33 286 1 A/A A/A 34 158 156 A/T A/T 35287 0 A/A A/A 36 118 133 T/T A/T 37 163 119 A/A A/T 38 273 10 A/T A/A 39132 148 T/T A/T 4 0 343 T/T T/T 40 143 177 T/T A/T 41 0 308 T/T T/T 42297 0 A/A A/A 43 117 130 T/T A/T 44 296 1 A/A A/A 45 276 0 A/A A/A 46140 134 A/T A/T 47 158 139 A/A A/T 48 0 304 T/T T/T 49 251 13 A/T A/A 5138 115 A/A A/T 50 142 162 T/T A/T 51 0 306 T/T T/T 52 249 21 A/T A/A 53111 170 T/T A/T 54 140 151 A/T A/T 55 102 217 T/T A/T 56 315 0 A/A A/A57 123 158 T/T A/T 58 146 168 T/T A/T 59 226 50 A/T A/A 6 309 0 A/A A/A60 122 133 T/T A/T 61 240 28 A/T A/A 62 132 124 A/T A/T 63 291 9 A/T A/A64 0 304 T/T T/T 65 273 0 A/A A/A 66 154 139 A/T A/T 67 145 153 A/T A/T68 110 163 T/T A/T 69 131 134 A/T A/T 7 186 127 A/A A/T 70 167 163 A/TA/T 71 238 26 A/T A/A 73 18 244 A/T T/T 75 130 129 A/T A/T 76 133 113A/A A/T 78 237 2 A/T A/A 79 0 278 T/T T/T 8 192 159 A/A A/T 80 153 131A/A A/T 81 25 229 A/T T/T 82 0 256 T/T T/T 83 152 142 A/T A/T 84 290 2A/T A/A 85 270 0 A/A A/A 86 0 242 T/T T/T 87 150 134 A/A A/T 89 169 117A/A A/T 9 271 1 A/A A/A 90 109 144 T/T A/T 91 261 12 A/T A/A 92 258 0A/A A/A 93 0 309 T/T T/T 94 116 146 T/T A/T 95 123 116 A/T A/T

The location of these SNPS is denoted using dbSNP version 132 andGRCH37/UCSC hg 19. The data for these polymorphisms was obtained in thesame data set as the aneuploidy data illustrated in FIGS. 10 and 11.Thus, a single assay demonstrated the ability to identify fetalaneuploidy, polymorphic differences between fetal and maternal loci, andthe actual SNP information for selected fetal loci in a single assay.

While this invention is satisfied by aspects in many different forms, asdescribed in detail in connection with preferred aspects of theinvention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific aspects illustrated anddescribed herein. Numerous variations may be made by persons skilled inthe art without departure from the spirit of the invention. The scope ofthe invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

What is claimed is:
 1. A method for simultaneous detection of thepresence or absence of a fetal copy number variation (CNV) of a genomicregion and presence or absence of one or more fetal polymorphisms in amaternal plasma or serum sample comprising fetal and maternal cell-freeDNA using a single assay, comprising the steps of: (a) hybridizing atleast 200 and less than 2000 first sets of two fixed sequenceoligonucleotides to the fetal and maternal cell-free DNA in the maternalplasma or serum sample in a first reaction, wherein the first sets oftwo fixed sequence oligonucleotides are complementary to a locus in afirst maternal and fetal genomic region from a first chromosome or aportion of the first chromosome without regard to polymorphisms in thelocus, wherein at least one of the fixed sequence oligonucleotidescomprises a universal primer region, wherein the two fixed sequenceoligonucleotides of each first set hybridize immediately adjacent toeach other, wherein the maternal plasma or serum sample comprises atleast 5% and less than 25% fetal DNA, and wherein the meltingtemperatures (T_(m)s) of first fixed sequence oligonucleotides of eachof the first sets of two fixed sequence oligonucleotides vary in rangeof two degrees centigrade; (b) hybridizing at least 200 and less than2000 second sets of two fixed sequence oligonucleotides to the fetal andmaternal cell-free DNA in the first reaction, wherein the second sets oftwo fixed sequence oligonucleotides are complementary to a locus in asecond maternal and fetal genomic region from a second chromosome or aportion of the second chromosome without regard to polymorphisms in thelocus, wherein at least one of the fixed sequence oligonucleotidescomprises a universal primer region, wherein the two fixed sequenceoligonucleotides of each second set hybridize immediately adjacent toeach other and wherein the T_(m)s of first fixed sequenceoligonucleotides of each of the second sets of two fixed sequenceoligonucleotides vary in a range of two degrees centigrade; (c)hybridizing a third set of two fixed sequence oligonucleotides to thefetal and maternal cell-free DNA in the first reaction, wherein thethird set of two fixed sequence oligonucleotides is complementary toregions on a maternal and fetal locus in a polymorphic region, whereinat least one of the fixed sequence oligonucleotides comprises auniversal primer region, and wherein the fixed sequence oligonucleotidesof the third set hybridize immediately adjacent to each other; (d)ligating in a multiplexed manner the hybridized oligonucleotides of thefirst, second and third sets of two fixed sequence oligonucleotides tocreate contiguous ligation products complementary to the loci in thefirst genomic region, the second genomic region, and the polymorphicregion; (e) amplifying in a multiplexed manner the contiguous ligationproducts using primers complementary to the universal primer regions ofthe first, second and third sets of two fixed sequence oligonucleotidesto create amplification products; (f) isolating the amplificationproducts to create isolated amplification products; (g) sequencing in amultiplexed, high throughput manner the isolated amplification productsan average of at least 100 times, wherein the sequenced isolatedamplification products are representative of the DNA content of thegenomic regions in the maternal sample; (h) detecting the presence orabsence of a fetal polymorphism in the polymorphic region; and (i)detecting the presence or absence of a fetal CNV by observing astatistical variation in the quantity of total sequence reads fromisolated amplification products of the first genomic region and thequantity of total sequence reads from isolated amplification products ofthe second genomic region.
 2. The method of claim 1, wherein theuniversal primer regions are used in sequence determination of theamplification products.
 3. The method of claim 1, wherein one or more ofthe sets of fixed sequence oligonucleotides comprise precircle probes.4. The method of claim 1, wherein the isolated amplification productsare further amplified to create identical copies of all or a portion ofthe amplification products prior to high throughput sequencedetermination.
 5. The method of claim 1, wherein the isolatedamplification products are further amplified to create identical copiesof molecules complementary to all or a portion of the amplificationproducts prior to high throughput sequence determination.
 6. The methodof claim 1, wherein the loci interrogated for copy number are differentfrom the loci interrogated for polymorphisms.
 7. The method of claim 1,wherein the first and second genomic regions are each single genes.
 8. Amethod for determining the likelihood of a fetal copy number variation(CNV) of a genomic region and presence or absence of one or more fetalpolymorphisms in a maternal plasma or serum sample comprising fetal andmaternal cell-free DNA using a single assay, comprising the steps of:(a) hybridizing at least 200 and less than 2000 first sets of two fixedsequence oligonucleotides to the fetal and maternal cell-free DNA in thematernal plasma or serum sample in a first reaction, wherein the firstsets of two fixed sequence oligonucleotides are complementary to aregion on a locus in a first maternal and fetal genomic region from afirst chromosome or a portion of the first chromosome without regard topolymorphisms in the locus, wherein at least one of the fixed sequenceoligonucleotides comprises a universal primer region, wherein the twofixed sequence oligonucleotides of each first set hybridize immediatelyadjacent to each other, wherein the maternal plasma or serum samplecomprises at least 5% and less than 25% fetal DNA, and wherein themelting temperatures (T_(m)s) of first fixed sequence oligonucleotidesof each of the first sets of two fixed sequence oligonucleotides vary inrange of two degrees centigrade; (b) hybridizing at least 200 and lessthan 2000 second sets of two fixed sequence oligonucleotides to thefetal and maternal cell-free DNA in the first reaction, wherein thesecond sets of fixed sequence oligonucleotides are complementary to aregion on a locus in a second maternal and fetal genomic region from asecond chromosome or a portion of the second chromosome without regardto polymorphisms in the locus, wherein at least one of the fixedsequence oligonucleotides comprises a universal primer region, whereinthe two fixed sequence oligonucleotides of each second set hybridizeimmediately adjacent to each other, and wherein the T_(m)s of firstfixed sequence oligonucleotides of each of the second sets of two fixedsequence oligonucleotides vary in a range of two degrees centigrade; (c)hybridizing a third set of two fixed sequence oligonucleotides to thefetal and maternal cell-free DNA in the first reaction, wherein thethird set of fixed sequence oligonucleotides is complementary to regionson a maternal and fetal locus in a polymorphic region, wherein at leastone of the fixed sequence oligonucleotides comprises a universal primerregion, and wherein the fixed sequence oligonucleotides of the third sethybridize immediately adjacent to each other; (d) ligating in amultiplexed manner the hybridized oligonucleotides to create contiguousligation products complementary to the loci in the first genomic region,the second genomic region, and the polymorphic region; (e) amplifying ina multiplexed manner the contiguous ligation products using primerscomplementary to the universal primer regions to create amplificationproducts; (f) isolating the amplification products to create isolatedamplification products; (g) sequencing in a multiplexed high throughputmanner the isolated amplification products an average at least 100 timesproducing total sequence reads; (h) detecting a likelihood of a fetalCNV using the quantified total sequence reads corresponding to the firstgenomic region and the quantified total sequence reads corresponding tothe second genomic region; wherein a likelihood of a fetal CNV isindicated if the quantified total sequence reads from the loci of firstand second genomic regions statistically vary; and (i) detecting thepresence or absence of a fetal polymorphism in the polymorphic regionfrom the sequence reads of the isolated amplification products from thelocus of the polymorphic region.
 9. The method of claim 8, wherein theuniversal primer regions are used in high throughput sequencedetermination of the isolated amplification products.
 10. The method ofclaim 8, wherein one or more of the sets of fixed sequenceoligonucleotides comprise a precircle probe.
 11. The method of claim 8,wherein the isolated amplification products are isolated as individualmolecules prior to high throughput sequence determination.
 12. Themethod of claim 11, wherein the individual isolated amplificationproducts are further amplified to create identical copies of all or aportion of the individual amplification products prior to highthroughput sequence determination.
 13. The method of claim 11, whereinthe individual isolated amplification products are further amplified tocreate identical copies of molecules complementary to all or a portionof the individual amplification products prior to high throughputsequence determination.
 14. The method of claim 8, wherein the lociinterrogated for copy number are different from the loci interrogatedfor polymorphisms.
 15. The method of claim 8, wherein the first andsecond genomic regions are each single genes.